Nuclear sulfated oxysterol, potent regulator of lipid homeostasis, for therapy of hypercholesterolemia, hypertriglycerides, fatty liver diseases, and atherosclerosis

ABSTRACT

The sulfated oxysterol 5-cholesten-3β, 25-diol 3-sulphate, a nuclear cholesterol metabolite that decreases lipid biosynthesis and increases cholesterol secretion and degradation, is provided as an agent to lower intracellular and serum cholesterol and/or triglycerides, and to prevent or treat lipid accumulation-associated inflammation and conditions associated with such inflammation. Methods which involve the use of this sulfated oxysterol to treat conditions associated with high cholesterol and/or high triglycerides and/or inflammation (e.g. hypercholesterolemia, hypertriglyceridemia, non-alcoholic fatty liver diseases, atherosclerosis, etc.) are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part (CIP) of U.S. patentapplication Ser. No. 11/739,330, filed Apr. 24, 2007, and claims benefitof U.S. provisional patent application 61/154,065, filed Feb. 20, 2009.U.S. Ser. No. 11/739,330 is a national stage CIP claiming benefit ofInternational patent application PCT/US2005/033874 filed on Sep. 21,2005, which in turn claims benefit of U.S. Provisional Application60/621,537 filed on Oct. 25, 2004. The complete contents of each ofthese applications are herein incorporated by reference.

STATEMENT OF FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made, in part, with government support under GrantNo.R01 HL078898 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

DESCRIPTION Background of the Invention

1. Field of the Invention

The invention generally relates to lipid-lowering therapies. Inparticular, the invention provides a nuclear cholesterol metabolite,5-cholesten-3β, 25-diol 3-sulphate, that decreases lipid biosynthesisand increases cholesterol secretion and degradation, and is thus usefulfor the treatment and prevention of hypercholesterolemia,hypertriglyceridemia, atherosclerosis, and conditions related tofat-accumulation and inflammation (e.g. nonalcoholic fatty liverdisease, NAFLD, and atherosclerosis).

2. Background of the Invention

Cholesterol is used by the body for the manufacture and repair of cellmembranes, and the synthesis of steroid hormones and vitamin D, and istransformed to bile acids in the liver. There are both exogenous andendogenous sources of cholesterol. The average American consumes about450 mg of cholesterol each day and produces an additional 500 to 1,000mg in the liver and other tissues. Another source is the 500 to 1,000 mgof biliary cholesterol that is secreted into the intestine daily; about50 percent is reabsorbed (enterohepatic circulation).

High serum lipid levels (hypercholesterolemia and hypertriglyceridemia)are associated with the accumulation of cholesterol in arterial walls,and can result in NAFLD and atherosclerosis. The plaques thatcharacterize atherosclerosis inhibit blood flow and promote clotformation, and can ultimately cause death or severe disability via heartattacks and/or stroke. A number of therapeutic agents for the treatmentof hyperlipidemia have been developed and are widely prescribed byphysicians. Unfortunately, only about 35% of patients are responsive tothe currently available therapies.

Nonalcoholic fatty liver disease (NAFLD) is the most common liverdisease in the United States. This condition is associated with obesity,type-II adult onset diabetes, sedentary lifestyle, and diets high infat. The earlier stage of NAFLD, fatty liver, is potentially reversiblewhen proper treatment steps are taken. However, left unchecked, it canprogress to inflammation of liver cells (nonalcoholic steatohepatitis,or NASH) which is much more difficult to treat. Without treatment, NASHcan result in irreversible scarring of liver tissue (steatonecrosis),with the potential to cause cirrhosis, liver failure, and liver cancer.

There is an ongoing need to develop agents and methodologies to decreaseintracellular and serum lipid levels, and to prevent or treat diseaseconditions involving the inflammation caused by elevated lipid levels.

SUMMARY OF THE INVENTION

The present invention provides a novel sulfated oxysterol,5-cholesten-3β, 25-diol 3-sulphate, with potent serum lipid loweringproperties. 5-Cholesten-3β, 25-diol 3-sulphate is a nuclear sterolmetabolite that decreases lipid biosynthesis and increases cholesterolsecretion and degradation (bile acid synthesis). The increase incholesterol degradation and decrease in lipid synthesis can lead tolower levels of intracellular and serum lipid levels. Thus, the sulfatedoxysterol is useful for preventing or treating diseases associated withelevated lipid levels, such as hypercholesterolemia,hypertriglyceridemia, gallstones, cholestatic liver disease,atherosclerosis, NAFLD, NASH, etc.

It is an object of this invention to provide a substantially purified5-cholesten-3β, 25-diol 3-sulphate having the following chemicalformula:

It is a further object of the invention to provide a lipid-loweringcomposition. The composition comprises 5-cholesten-3β, 25-diol3-sulphate, and a pharmaceutically acceptable carrier.

It is a further object of the invention to provide a method for loweringserum lipids levels (such as cholesterol and triglyceride levels) in apatient in need thereof. The method comprises the step of administering5-cholesten-3β, 25-diol 3-sulphate to the patient in an amountsufficient to lower serum lipid levels, e.g. cholesterol andtriglyceride levels, in the patient.

The invention further provides a method to treat or prevent pathologicalconditions associated with high serum lipids (e.g. cholesterol andtriglyceride) levels in a patient in need thereof. The method comprisesthe step of administering 5-cholesten-3β, 25-diol 3-sulphate to thepatient in an amount sufficient to lower serum lipid levels in thepatient, and to prevent or treat the pathological condition. Thepathological condition is, for example, hypercholesterolemia,hypertriglyceridemia, atherosclerosis, or NAFLD.

The invention further provides a method of preventing or treatinginflammation caused by or associated with lipid accumulation, orconditions or diseases associated with lipid accumulated-inflammation ina patient in need thereof. The method comprises the step ofadministering 5-cholesten-3β, 25-diol 3-sulphate to the patient in anamount sufficient to prevent or treat the inflammation or the conditionassociated with inflammation in the patient. In one embodiment, theconditions associated with inflammation are non-alcoholic fat liverdiseases and atherosclerosis.

The invention also provides a method of decreasing lipid synthesis in apatient in need thereof. The method comprises the step of administering5-cholesten-3β, 25-diol 3-sulphate to the patient in an amountsufficient to decrease lipid synthesis in the patient.

The invention further provides a method of increasing cholesterolsecretion or degradation in cells. The method comprises the step ofincreasing a level of 5-cholesten-3β, 25-diol 3-sulphate in the cells.The method may be carried out by exposing the cells to 5-cholesten-3β,25-diol 3-sulphate.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Subcellular fractionation protocol. For details see“Experimental Procedures” in Example 1.

FIGS. 2A-C. Effects of overexpression of StarD1 on Cyp7A1 mRNAexpression in primary rat hepatocytes. At day five following infectionof virus control, virus encoding StarD1, and CYP27A1 as indicated, cellswere harvested and total RNAs were extracted. To each lane, 20 μg oftotal RNA was loaded. Specific Cyp7A1 mRNAs (Panel A) were determined byNorthern blot analysis. Cyclophilin was used as control (Panel B). Theexpressions of Cyp7A1 mRNAs were increased by 6-fold (n=3) followingoverexpression of StarD1 and 2.5 fold following CYP27A1 as indicated(Panel C).

FIG. 3. Thin layer chromatographic analysis of the chloroformextractable cholesterol derivatives. Rat primary hepatocytes wereinfected with the indicated viruses. Forty-eight hrs later cells wereharvested and nuclear lipids extracted and analyzed as explained under“Experimental Procedures”.

FIGS. 4A and B. Phase distribution of [¹⁴C]cholesterol derivatives innuclei of primary rat hepatocytes following overexpression of StarD1 andCYP7A1. Rat primary hepatocytes were infected with the indicatedviruses. Forty-eight hrs later cells were harvested and subcellularfractions prepared. Fractions E and F were processed for lipid analysisas explained under Experimental Procedures”. A, Nuclear inner membrane(Fraction E). B, Nuclear digests (Fraction F).

FIGS. 5A-C. HPLC analysis of [¹⁴C]cholesterol derivatives in the nuclearfraction (Fraction D) and non-nuclear fraction (Fraction A). Twenty-fourhrs following the indicated recombinant adenovirus infection, cells wereharvested and nuclear and non-nuclear fractions (Fractions A and D) wereisolated, extracted by the Folch method and the methanol/water phaseanalyzed. A, nuclear extracts (Fraction D) 195 nm profiles. B, nuclearextracts (Fraction D)¹⁴C profiles. C, non-nuclear extracts (FractionA)¹⁴C profiles. In each case, nuclear methanol/water extracts of theequivalent of 5×10⁶ cells were loaded.

FIGS. 6A-F. HPLC analysis of the cholesterol derivatives extracted fromthe nuclei, mitochondria, and culture media. Rat primary hepatocyteswere infected with the StarD1 adenovirus and two hrs later[¹⁴C]cholesterol was added to the media. Twenty-four hrs following,cells and culture media were harvested. Nuclei and mitochondria wereisolated as described under “Experimental Procedures”. Total lipids wereextracted from the nuclei, mitochondria, and culture media by Folchpartitioning into methanol phase, and analyzed by HPLC as described in“Experimental Procedures”. A-C, 195 nm profiles. D-F, radioactivityprofiles.

FIGS. 7A-D. Characterization of the nuclear oxysterol by enzymaticdigestion followed by HPLC and TLC. Nuclear [¹⁴C]oxysterol derivativeswere isolated from StarD1 overexpressing rat primary hepatocytes anddigested with 1 mg/ml of sulfatase in acetic acid buffer, pH 5.0,overnight. Total lipids were extracted with chloroform/methanol andseparated by Folch partitioning. The products in the chloroform andmethanol/water phase were analyzed by HPLC using a mixture of 965 mlhexane, 25 ml isopropanol, and 10 ml acetic acid as mobile phase, 1.3ml/min flow rate and ¹⁴C quantified. A, HPLC elution profile of thesulfatase digestion products. B, HPLC elution profile of[¹⁴C]27-hydroxycholesterol. C, HPLC elution profile of[¹⁴C]25-hydroxycholesterol. D, products from the chloroform phase werefurther analyzed by TLC using a mixture of toluene:ethyl acetate (2:3)as developing solvent. 27-C represents 27-hydroxycholesterol; P,sulfatase digestion products of the purified nuclear oxysterols; 25-C,25-hydroxycholesterol.

FIGS. 8A-C. Characterization of nuclear oxysterol by negative ion-triplequadruple mass spectrometry (LC/MS/MS). (A) A selected ion chromatogramof mass ion at m/z 481; (B) the Q1 full scan spectrum; (C) product scanspectrum of m/z 481. The amu represents atomic mass units, and cps,counts per second.

FIGS. 9A-B. Effect of the nuclear oxysterol on cholesterol uptake andbile acid biosynthesis. Rat primary hepatocyes were treated with nuclearextracts (methanol/water phase) (A and B) or purified nuclear oxysteroldissolved in control nuclear extract (C) 24 hrs after plating them. Then[¹⁴C]cholesterol was added as described in FIG. 1. Culture media werethen harvested at 0, 6, 12, and 24 hrs and radioactivity quantified (A).Bile acid synthesis rates (B and C) were measured as the conversion of[¹⁴C]cholesterol into methanol/water extractable counts as described in(9).

FIG. 10. ABCA1 (A1), ABCG1 (G1), LDL receptor (LDLR), ABCG5 (G5), andABCG8 (G8) gene expression in primary mouse hepatocytes followingaddition of the purified nuclear oxysterol. At 24 hrs following theaddition, cells were harvested and total RNAs were extracted and geneexpression levels were quantitated by real time RT-PCR. β-Actin mRNA wasused as total mRNA internal standard. The gene expression levels incells with StarD1 overexpression were compared with those in controlcells. Ten μg of total RNA was used for cDNA preparation (RT) and 10 ngof cDNA was used for PCR. The expression levels were normalized toβ-actin.

FIG. 11. Biosynthesis pathway of nuclear sulfated oxysterol. In thepresence of StarD1 protein, cholesterol is delivered into mitochondriawhere 25-hydroxylase (25-OHLase) and hydroxylcholesterol sulfatetransferase 2b (HST2b) locate, and converted to be 25-hydroxycholesterol3-sulfate. This sulfated oxysterol translocates to nucleus and regulatesgene expressions involved in cholesterol metabolism.

FIGS. 12A-D. (A) addition of sulfate group onto 3β-position of25-hydroxycholesterol for the synthesis of the novel nuclear oxysterolby incubation with sulfur trioxide triethyl amine complex; (B) massspectrophometric analysis of the product after incubation with thesulfur trioxide and purified by HPLC. Mass ion, m/z 481, represents25-hydroxycholesterol (M.W. 482)+Sulfate group (M.W.80); (C) nuclearmagnetic resonance (NMR) analysis of the 25-hydroxycholesterol 3-sulfateas starting material for the synthesis. The chemical shift of the protonat C3 in the molecule can be seen at 3.35 ppm; and (D) NMR analysis ofthe product shows the proton at C3 in the molecule has been shifted to4.12 ppm from 3.35 ppm in its original compound.

FIGS. 13A-C. TLC and HPLC analysis of the newly synthesized[¹⁴C]cholesterol. After incubation of the 25HC3S-treated cells with[1-¹⁴C]acetate for 2 hrs, the cells were harvested. The total neutrallipids were extracted with chloroform/methanol and partitioned intochloroform phase. The [¹⁴C]-acetate derivatives were analyzed by thinlayer chromatography (TLC) and HPLC. A. TLC analysis of the[¹⁴C]-acetate derivatives: the chloroform phase extracts of theequivalent of 5×10⁶ cells were loaded onto each lane, separated bydeveloping system of tuluene:acetyl acetate, and visualized by ImagineReader. B. HPLC analysis of the [¹⁴C]-acetate derivatives: thechloroform phase extracts of the equivalent of 5×10⁶ cells were loadedonto silica gel column. The effluents were collected, 0.5 min/fraction.The radioactivities in each fraction were determined by ScintillationCounting. C. A summary of three experiments of HPLC analysis. Each barrepresents the mean of three experiments±standard deviation.

FIGS. 14A-H. HPLC analysis of cholesterol levels in microsomalfractions. The total lipids were extracted from 25HC3S-treated (leftpanels) or 25HC- (right panels) treated HepG2 cells. α,β-Unsaturatedketones were generated by incubating the extracted sterols withcholesterol oxidase and were analyzed by normal phase HPLC: Panels A-C:the lipids from the cells treated with 0, 3, and 6 μM of 25HC3S; D. Asummary of a series experiments, the lipids from the cells treated with0, 3, 6, 12, and 25 μM of 25HC3S. Panels E-G: the lipids from the cellstreated with 0, 3, and 6 μM of 25HC; D. A summary of a seriesexperiments, the lipids from the cells treated with 0, 3, 6, 12, and 25μM of 25HC. The data represent a typical result from one of threeindependent experiments.

FIGS. 15A-D. 25HC3S regulates HMGR mRNA Expression. Real time RT-PCRanalysis of HMG CoA R reductase expression in HepG2 cells (Panels A andB). Western blot analysis of protein levels of HMG CoA reductase (PanelC). Total RNAs were purified using SV Total RNA Isolation Kit (Promega)from the cells treated with 25HC3S at concentrations as indicated (A)and incubation at 12 μM of 25HC3S for the different times (effect on HMGCoA mRNA) (Panel B). Two μg of total RNA was used for cDNA preparation(RT) and performed as manufacture recommended (Invitrogen), and 10 ng ofcDNA was used for real time PCR. The expression levels were normalizedto GAPDH. The total extracted protein, 100 was loaded in each well foreach condition as indicated in panel C. The bands of HMG CoA reductase(HMGR) were qualitated by laser density scanning. The data from threeexperiments were summarized in Panel D. Each value represents mean ofthree experiments±standard derivation.

FIGS. 16A-C. Effects of 25HC and 25HC3S on the levels of HMG CoAreductase mRNA in hepatocytes. Total RNA was extracted from the HepG2cells cultured either in 10% FBS media (Panel A) or in 10%lipid-depleted sera (Panel B) and treated with either 25HC or 25HC3S asindicated. The real time RT-PCR analysis was performed as described inFIG. 16. The data represent a typical result from one of threeindependent experiments. The 25HC or 25HC3S affects the HMG CoAreductase expression in primary human hepatocytes (Panel C). The datarepresent a typical result from one of three independent experiments.

FIGS. 17A-E. HPLC analysis of 25HC in PHH cells treated with 25HC. Thetotal lipids were extracted from 25HC-treated PHH cells. α,β-Unsaturatedketones were generated by incubating the extracted sterols withcholesterol oxidase and were analyzed by normal phase HPLC: Panels A-E:the lipids from the cells treated with 0, 3, 6, 12, 25 μM of 25HC asindicated; The data represent a typical result from two independentexperiments.

FIGS. 18A-D. Western blot analysis of SREBPs activation following 25HC3Sor 25HC treatment in HepG2 cells. Total proteins were extracted fromHepG2 cells treated with 25HC in ethanol (0.1%) (Panel A and C) or25HC3S in DMSO (0.1%) (Panel B and D), cultured in the media in absence(Panels A and B) or presence (Panels C and D) of mevinolin (50 mM) andmevalonate (0.5 mM). SREBP-1 and SREBP-2 protein levels in the cellswere determined by Western blot analysis. The extracted protein (100 μg)was loaded onto each lane for each condition as indicated. The datarepresent a typical result from one of three independent experiments.The trends of the protein levels are highly reproducible.

FIG. 19. Relative levels of cholesterol in HepG2 microsomal fractions (yaxis) vs concentration of 25HC3S (x axis).

FIG. 20. Lipids in mouse sera after injection of 25HC3S.

FIGS. 21A and B. Effects of 25HC3S and 25HC on PPARγ protein levels inTHP-1 macrophages. A, THP-1 macrophages were treated with 25HC3S (toppanel) or 25HC (middle panel) at different concentration as indicatedfor 4 hrs. The nuclear PPARγ levels were analyzed by Western blot. B,The macrophages were pre-incubated with 1 μM of T0070907 for 2 hrs andtreated with 25HC3S (tip panel) or preincubated with 1 μM ofrosiglitazone for 2 hrs; and treated with 25HC for 4 hrs (middle panel).Expression levels were normalized to Lamin as shown in the bottompanels. The data represent one typical result out of three experiments.

FIGS. 22A-E. 25HC3S enhances nuclear PPRE transcriptional activities andinhibits LPS-induced TNF-α and IL-1B expressions and releases. THP-1macrophages were treated with the indicated concentrations of 25HC3S or2 μM of rosiglitazone for 4 hrs. A, The nuclear proteins were extractedand PPRE transcriptional activities were determined by the ELISA. Rrepresents rosiglitazone. Reporter gene activity assays were determinedin H441 cells following co-transfection with promoter reporter gene andexpression of plasmid pCMX-PPARγ:25HC3S (B) or rosiglitazone (C) with orwithout 1 μM of T0070907 was added and incubated for another 24 hrs.Luciferase activities were determined. For competitive assays, the cellswere preincubated with T0070907 for 1 hr and incubated with 25HC3S atthe different concentrations as indicated. After 24 hrs, the cells wereincubated with LPS for 3 hrs. The expression of mRNA for TNFα and IL-1βwas determined by real-time RT-PCR (D). The released TNFα and IL-1Bconcentrations in the media following the addition of LPS were measuredby ELISA respectively (E). Data represent means±SD (n=3). The symbol *represents significant difference (p<0.05).

FIGS. 23A and B. 25HC3S-mediated suppression of NFκB activation isPPARγ-dependent. A, THP-1 macrophages were treated with 25HC3S or 25HCfor 24 hrs followed by addition or no addition of TNFα for another 3hrs. The nuclear NFκB levels were analyzed by Western blot. The datarepresent one typical result out of three experiments. B, NFκBactivation is PPARγ dependent. H441 cells were transfected with pNFκBdependent reporter gene-Luc, treated with or without T0070907 for 1 hr,and incubated with 25HC3S at indicated concentration for 24 hrs. Thereporter gene expression was induced by incubating with 10 ng/ml of TNFαfor 3 hrs. The left figure represents cells transfected with pNFκBdependent reporter gene-Luc plasmid alone; the right figure, representscells co-transfected with PPARγ expression plasmid. The symbol *represents significant difference (p<0.05).

FIGS. 24A-E. 25HC3S failed to suppress LPS induced TNFα, IL-1β and NFκB,and TNFα induced IκB mRNA expression in the PPARγ knock downmacrophages. Following expression of PPARγ-specific siRNAs in themacrophages treated with or without 12 μM of 25HC3S for 48 hrs, nuclearPPARγ protein levels were determined by Western bolt analysis as shownin A. The mRNA levels of IκB and NFκB (B), TNFα (C), IL-1 (D), and NFκB(E) were determined by real time RT-PCR analysis. The values representmeans±SD (n=3). The symbol * represents statistically significance(p<0.05).

FIGS. 25A-C. Effects of 25HC3S on expression of IκB mRNA and proteinlevels in THP-1 macrophages. Effects of 25HC3S and 25HC on IκB proteinlevels were analyzed by Western blot following the treatment for 24 hrs(A and B) and mRNA levels were determined by real time RT-PCR followingthe treatment for 6 hrs (C). The Western blot data represents one ofthree experiments.

FIG. 26. Role of 25HC3S (oxysterol sulfation) in inflammatory responsein THP-1 derived macrophages. 25-Hydroxycholesterol (25HC) is sulfatedby a sulfotransferase SULT2B1 to form 25HC3S, and this reaction can bereversed by steroid sulfatase (STS). 25HC3S activates PPARγ and theactivated PPARγ enters the nucleus where it up-regulates IκB expressionand suppresses TNFα expression. As an inactive form, NFκB is bound bymembers of IκBs and sequestered in the cytoplasm. When TNFα levels areincreased, it removes IκBs from NFκB by ubiquitination and degradation,and subsequently activates NFκB. The free active NFκB enters nuclei forstimulation of inflammatory response. Thus, 25HC3S repressesinflammatory response by activating of PPARγ and subsequentlysuppressing TNFα and stimulating IκB expression. However, its precursor25HC inactivates PPARγ and increases IκB degradation, which favorspro-inflammatory responses.

FIGS. 27A-F. 25HC3S increases nuclear translocation of PPARγ in THP-1macrophages. Confocal microscopy analysis of intracellular translocationof PPARγ protein distribution. The nuclear marker was stained with DAP1and PPARγ protein was detected by anti-PPARγ immunofluorescence. Forevery figure, the top left panel shows nuclear staining; the top rightpanel shows the localization of PPARγ with the monoclonal antibody; thebottom right panel shows a differential interference contrast (DIC)image; the bottom right panel shows the merged image obtained bysuperimposing the three images mentioned above. Macrophages were treatedwith vehicle DMSO under 100× magnification (A) and 60× (B); treated with25 μM of 25HC3S, 100× (D) and 60× (E); treated with 1 μM of T0070907 (C;preincubated with 1 of T0070907 for 2 hrs and cultured with 25HC3S for 4hrs (F). The data represent one of three separate experiments. Scalebar=10 μm.

FIGS. 28A and B. Addition of 25HC3S suppresses LPS-induced IL-1β (A) andTNFα (B) expression in macrophages. To study the effect of 25HC3S onLPS-induced TNFα and IL-1β expression and release, macrophages weretreated with 1 μg/ml of LPS and different concentrations of 25HC3S and25HC as indicated for 25 hours; mRNA levels of TNFα and IL-1β weredetermined by real time RT-PCR. The results represent mean±SD. Additionof LPS dramatically increased IL-1β mRNA levels by 55-fold. Addition of25HC3S significantly suppressed this increase, while 25HC significantlyincreased levels still further (A). Both 25HC3S and 25HC repressedLPS-induced TNFα mRNA levels as shown in B.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

Disordered lipid metabolism and inflammatory responses play importantroles in the pathogenesis of many fatal diseases, includingatherosclerosis and nonalcoholic fatty liver diseases (NAFLD). The“two-hit” theory is widely accepted for explaining the occurrence anddevelopment of the diseases. The “first hit” is the initialintracellular lipid accumulation, whilst the “second hit” isinflammation and injury. Therefore, decreasing intracellular lipidlevels and repressing the inflammatory response is the key to developinga new generation of therapeutic agents against these devastatingdiseases.

Recently, we have discovered the novel nuclear regulatory oxysterol(5-cholesten-3β, 25-diol 3-sulphate), and elucidated its metabolicpathway, and studied its role in lipid metabolism and inflammation. Theoxysterol down-regulates key enzymes involved in lipid biosynthesis andsubsequently decreases intracellular lipid levels by blocking theactivation of liver oxysterol receptor (LXR)/sterol regulatory elementbinding protein signaling pathway. On the other hand, oxysteroldecreases pro-inflammatory cytokine expression and secretion, andrepresses inflammatory response by activating peroxisome proliferationactivator receptor gamma (PPARγ) and PPARγ/IκB/NFκB signaling pathway inmacrophages and hepatocytes. A large body of experimental evidence hasdemonstrated that the oxysterol plays an important role in both “hits”.

Most importantly, in vivo experimental studies showed that only oneadministration of the oxysterol decreases serum cholesterol andtriglyceride by 20˜40% and significantly increases HDL. Small moleculePPARγ agonists have been used for therapy of diabetes for a couple ofdecades and small molecule LXR agonists have been tested for therapy ofhypercholesterolemia for couple of years. However, these agonistsinevitably carry serious side effects. The new oxysterol, a naturallyoccurring PPARγ agonist and LXRs antagonist, has been shown to inhibitcholesterol and lipid biosynthesis in both hepatocytes and macrophagesvia LXR/SREBP and repress inflammatory responses via PPARγ/IκB/NFκBsignaling pathway. Therefore, this naturally occurring oxysterol servesas a new class of medication for the treatment and the prevention ofatherosclerosis, fatty liver, diabetes, and inflammatory diseases.

Both in vivo and in vitro data demonstrated that nuclear oxysterolincreases nuclear PPARγ and decreases nuclear LXR levels in hepatocytesand macrophages. The nuclear oxysterol can decrease not onlyintracellular lipid levels but also repress the inflammatory response.Therefore the therapeutic concept of nuclear oxysterol represents a newapproach to treating and preventing fatal diseases such asatherosclerosis and nonalcoholic fatty liver diseases.

The present invention is based on the discovery of 5-cholesten-3β,25-diol 3-sulphate (25HC3S), a novel sulfated oxysterol with potentlipid (e.g cholesterol and triglyceride) lowering properties. Thechemical structure of the sulfated oxysterol is as follows:

This sulfated oxysterol is a nuclear cholesterol metabolite thatdecreases cholesterol and triglyceride levels in serum. The compoundincreases cholesterol secretion by increasing expression of cholesteroltransporters in hepatocytes. The increase in cholesterol secretion anddegradation ultimately leads to lower levels of serum cholesterol andtriglycerides. Without being bound by theory, it appears that thesulfated oxysterol is made in the mitochondrion and translocates to thenucleus of the cell, where it acts to up-regulate genes involved incholesterol and triglyceride metabolism. 5-cholesten-3β, 25-diol3-sulphate is thus useful for preventing or treating diseases associatedwith elevated cholesterol (hypercholesterolemia) and triglycerides(hypertriglyceridemia), such as hyperlipidemia, atherosclerosis,coronary heart disease, stroke, non-alcoholic fatty liver diseases, etc.This sulfated oxysterol is especially suitable for in vivo use becauseit is an authentic natural compound that is biosynthesized in vivo byhydroxylation and sulfation of cholesterol. Thus, 5-cholesten-3β,25-diol 3-sulphate should have few or no toxic side effects whenadministered to patients. In addition, the invention provides methods ofpreparing and administering 5-cholesten-3β, 25-diol 3-sulphate.

Example 6 shows that 5-cholesten-3β, 25-diol 3-sulphate also inhibitsinflammation. Without being bound by theory, it appears that 25HC3 Sacts in macrophages as a PPARγ activator, and suppresses inflammatoryresponses via PPARγ/IκB/NFκB signaling pathway. 25HC3S appears toattenuate the inflammatory response by increasing IκB expression anddecreasing IκB ubiquitination and degradation, thus exerting its effectthrough the PPARγ/IκB/NFB signaling pathway. As such, this compound isalso useful for treating conditions that are caused or exacerbated byinflammation, particularly inflammation caused by lipid accumulation incells, such as arthrosclerosis and fatty liver diseases.

The compound of the invention will be provided in a substantiallypurified form for use in the methods of the invention. By “substantiallypurified” we mean that the sulfated oxysterol is provided in a form thatis at least about 75%, preferably at least about 80%, more preferably atleast about 90%, and most preferably at least about 95% or more freefrom other chemical species, e.g. other macromolecules such as proteinsor peptides, nucleic acids, lipids, and other cholesterol-relatedspecies (e.g. other cholesterol derivatives such as cholesterolmetabolites, chemically modified forms of cholesterol such as variousother hydroxylated cholesterol species, etc.). In one embodiment of theinvention, the sulfated oxysterol of the invention may be isolated andpurified from living cells. One embodiment of this method is describedin Example 1 in the Examples section below. However, those of skill inthe art will recognize that in order to generate larger quantities ofthe sulfated oxysterol, the compound may also be synthesized, either bysynthetic chemical means, or by methods which involve the use ofrecombinant DNA technology (e.g. by using cloned enzymes to carry outsuitable modifications of cholesterol). An exemplary synthesis schemefor 5-cholesten-3β, 25-diol 3-sulphate is as follows: A mixture of25-hydroxycholesterol (0.1 mmol) and sulfur trioxide triethyl aminecomplex (0.12 mmol) in dry pyridine was stirred at room temperature for2 hrs. The solvent was evaporated at 40° C. under nitrogen stream, andthe pellets were dissolved in 500 ul of alkalined CH₃OH, pH 8.0. Thesodium 5-cholesten-3b, 25-diol 3-sulfate was purified by flashchromatography to afford the product as a white solid.

The methods of the invention are useful for the treatment or preventionof conditions associated with high levels of cholesterol andtriglyceride (hyperlipidemia). Such conditions may be either caused orexacerbated by high cholesterol and triglyceride, and include but arenot limited to hyperlipidemia, atherosclerosis, heart disease, stroke,Alzheimer's, gallstone diseases, cholestatic liver diseases,non-alcoholic fatty liver disease (NAFLD), etc. As used herein NAFLD isintended to encompass early stages of the diseases (e.g. fataccumulation in liver cells), stages in which the fatty liver cellsbecome inflamed (e.g. NASH or hepatitis), etc. The compound is alsouseful to treat inflammation or conditions caused or exacerbated byinflammation, for example, atherosclerosis, inflammatory bowel diseases,and diabetes (e.g. type 2 adult onset diabetes). By “treat” we mean thata disease condition has already developed, and the methods of theinvention are used to ameliorate symptoms of the disease condition,either to stop or decrease progression of the disease, or to reversesymptoms of the disease, either partially or fully. Alternatively, by“prevent” we mean that the compounds of the present invention may beadministered to patients prophylactically prior to the development ofdisease symptoms, e.g. to one who has high cholesterol but has not yetdeveloped atherosclerosis, or to one who does not yet have highcholesterol but is at high risk for developing high cholesterol (e.g. asdetermined by genetic factors, family history, etc.); or to a patientdiagnosed with fatty liver disease who is at risk of developing NASH orother conditions associated with high lipids, fat accumulation in cells,etc.

Those of skill in the art will recognize that the phrases “high lipidlevel”, “high cholesterol level” and “high triglyceride level” generallyrelate to cholesterol levels in serum in the range of about 200 mg/dl ormore, and triglyceride levels in serum greater than about 150 mg/dl. Adetermination of “high cholesterol” or “high triglyceride” is typicallymade by a health professional such as a physician, and the establishedmeaning of “high” levels may vary somewhat from professional toprofessional. Further, the precise definition may vary somewhatdepending on the state of the art, e.g. on findings from studies whichinvestigate the relationship between lipid levels and diseases.Nevertheless, those of skill in the art will be able to identifysuitable candidates for administration of the sulfated oxysterol of thepresent invention. By “lowering lipid levels” or “lowering triglyceridelevels” or “lowering cholesterol levels” we mean that the level of freeserum lipid/cholesterol/triglyceride in a patient is decreased by atleast about 10% to 30%, and preferably at least about 30 to 50%, andmore preferably at least about 50 to 70%, and most preferably at leastabout 70 to about 100%, or more, in comparison to the level of lipid inthe patient prior to administration of the sulfated oxysterol.Alternatively, the extent of the decrease may be determined bycomparison to similar untreated control individuals to whom the compoundis not administered. Those of skill in the art are familiar with suchdeterminations, e.g. the use of controls, or the measurement of lipidlevels in the blood before and after administration of an agent thatlowers lipids.

Implementation of the methods of the invention will generally involveidentifying patients suffering from or at risk for developing conditionsassociated with high lipids, and administering the compound of thepresent invention in an acceptable form by an appropriate route. Theexact dosage to be administered may vary depending on the age, gender,weight, and overall health status of the individual patient, as well asthe precise etiology of the disease. However, in general foradministration in mammals (e.g. humans), dosages in the range of fromabout 0.1 to about 100 μg or more of compound per kg of body weight per24 hr., and preferably about 0.1 to about 50 μg of compound per kg ofbody weight per 24 hr., and more preferably about 0.1 to about 10 μg ofcompound per kg of body weight per 24 hr. are effective.

Administration may be oral or parenteral, including intravenously,intramuscularly, subcutaneously, intradermal injection, intraperitonealinjection, etc., or by other routes (e.g. transdermal, sublingual, oral,rectal and buccal delivery, inhalation of an aerosol, etc.). In apreferred embodiment, administration is oral. Further, administration ofthe compound may be carried out as a single mode of therapy, or inconjunction with other therapies, e.g. other cholesterol lowering drugs,exercise and diet regimens, etc.

The compounds may be administered in the pure form or in apharmaceutically acceptable formulation including suitable elixirs,binders, and the like (generally referred to a “carriers”) or aspharmaceutically acceptable salts (e.g. alkali metal salts such assodium, potassium, calcium or lithium salts, ammonium, etc.) or othercomplexes. It should be understood that the pharmaceutically acceptableformulations include liquid and solid materials conventionally utilizedto prepare both injectable dosage forms and solid dosage forms such astablets and capsules and aerosolized dosage forms. In addition, thecompounds may be formulated with aqueous or oil based vehicles. Watermay be used as the carrier for the preparation of compositions (e.g.injectable compositions), which may also include conventional buffersand agents to render the composition isotonic. Other potential additivesand other materials (preferably those which are generally regarded assafe [GRAS]) include: colorants; flavorings; surfactants (TWEEN, oleicacid, etc.); solvents, stabilizers, elixirs, and binders or encapsulants(lactose, liposomes, etc). Solid diluents and excipients includelactose, starch, conventional disintegrating agents, coatings and thelike. Preservatives such as methyl paraben or benzalkium chloride mayalso be used. Depending on the formulation, it is expected that theactive composition will consist of about 1% to about 99% of thecomposition and the vehicular “carrier” will constitute about 1% toabout 99% of the composition. The pharmaceutical compositions of thepresent invention may include any suitable pharmaceutically acceptableadditives or adjuncts to the extent that they do not hinder or interferewith the therapeutic effect of the sulfated oxysterol.

The administration of the compound of the present invention may beintermittent, or at a gradual or continuous, constant or controlled rateto a patient. In addition, the time of day and the number of times perday that the pharmaceutical formulation is administered may vary and arebest determined by a skilled practitioner such as a physician. Further,the effective dose can vary depending upon factors such as the mode ofdelivery, gender, age, and other conditions of the patient, as well asthe extent or progression of the disease condition being treated. Thecompounds may be provided alone, or in combination with othermedications or treatment modalities.

In addition, the compound of the invention may also be used for researchpurposes.

EXAMPLES Example 1

Sterol ligands play key roles in the maintenance of lipid homeostasis.The present study has identified a novel regulatory nuclear sulfatedoxysterol, which is generated in mitochondria, translocates to thenucleus, and upregulates the rate of bile acid synthesis. At forty-eighthrs after infection with recombinant adenovirus encoding a mitochondriacholesterol transport protein (StarD1) in primary rat hepatocytes, bileacid synthesis increased by 5-fold. Concurrently, [¹⁴C] oxysterolderivatives with retention time at 11.50 min in HPLC elution profile wasdramatically increased both in the mitochondria and in the nucleus, butnot in culture media. The oxysterol product could be extracted into thechloroform phase from the methanol/water phase after sulfatasetreatment, and had the same physical properties as25-hydroxycholesterol. LC/MS/MS analysis showed the nuclear oxysterolwith a molecular ion, m/z 481, in the Q1 full scan spectrum, and thepresence of fragment ions at m/z 59, 80, 97, and 123 in its product scanspectrum. Thus, the nuclear oxyserol derivative can be characterized as5-cholesten-3β, 25-diol 3-sulphate. The addition of nuclear extract fromthe cells overexpressing StarD1 or the addition of the purifiedoxysterol to primary rat hepatocytes significantly increased the ratesof bile acid synthesis (>3.5 fold), suggesting this oxysterol derivativeis an active regulator. These results provide evidence for a newregulatory pathway by which a novel potent regulatory nuclear sulfatedoxysterol is generated in mitochondria, translocates to the nucleus, andupregulates bile acid synthesis.

Introduction

The biotransformation of cholesterol to primary bile acids occurs viatwo main pathways in hepatocytes (1). The “neutral” pathway isconsidered to be the major pathway at least in humans and rats (2). Inthis pathway the sterol nucleus is modified before the side-chain,beginning with hydroxylation of cholesterol at the 7α position. Thisreaction is catalyzed by cholesterol 7α-hydroxylase (CYP7A1), the firstand rate-limiting step of this pathway. The ability to lower plasmacholesterol levels via the pharmacological control of CYP7A1 expressionrepresents a therapeutic approach that has been in use for the last 30years and is still of intense research interest. Multiple negative andpositive modulators of CYP7A1 transcription have been identified both intissue culture systems and in vivo (3) and many of these modulators areoxysterols, such as hydroxy-cholesterol molecules and bile acids. Theyfunction by activating nuclear receptors, such as liver X receptor (LXR)and farnesoid X receptor (FXR), which in turn regulate the expression ofregulatory genes involved in bile acid biosynthesis, such as CYP7A1 andsterol 12α-hydroxylase (CYP8B1), the enzyme specific for cholic acidsynthesis. Oxysterols are also key regulatory molecules for theexpression of many other genes involved in the homeostasis ofcholesterol, and other lipids, such as 3-hydroxy-3-methyl-glutarylcoenzyme A (HMG-CoA) reductase, low density lipoprotein (LDL) receptor,some ATP-binding cassette transporters, like the ABCA1 and ABCG8, andmany others. They function by modulating the activity of either nuclearreceptors or other transcriptional factors, such as the sterolregulatory binding proteins (SREBPs) (4-6). Thus, characterizingendogenous synthesized oxysterols and their mechanism of action iscritical for a better understanding of lipid homeostasis. The initialstep in the “acidic” pathway is catalyzed by the enzyme mitochondrialsterol 27-hydroxylase (CYP27A1). The oxysterol intermediates of the“acidic” pathway such as 25-, or 27-hydroxycholesterol have been shownin vitro to be potent regulators in cholesterol homeostasis (7).Increased CYP27A1 activity in peripheral tissues may both down-regulatecholesterol synthesis through the SREBP pathway, and enhance the effluxof cholesterol and its elimination via LXR (8). However, thephysiological and authentic in vivo LXR ligand is unknown (9).

We have recently found that overexpression of steroidogenic acuteregulatory protein (StarD1), a protein which facilitates cholesteroltransport to the mitochondria, dramatically increases cholesteroltransport into mitochondria, the hydroxylation of cholesterol tooxysterol, and cholesterol catabolism to bile acids both in primaryhepatocytes in culture and in vivo (10;11). This suggests thatcholesterol delivery to the mitochondria, where the enzyme CYP27A1 islocalized, is the rate-determining step for bile acid synthesis via theacidic pathway. Subsequently, StarD1 was found in isolated hepatocytes(12). Overexpression of StarD1 in vivo increases bile acid synthesis notonly to the same level as overexpression of CYP7A1, but also produces asimilar composition of bile acids in mouse bile in vivo (11). Thus, itis reasonable to hypothesize that potent oxysterol molecule(s) might begenerated in the mitochondria, thereby regulating bile acid synthesis,and playing an important role in maintenance of intracellularcholesterol homeostasis.

To test this hypothesis, a recombinant adenovirus encoding StarD1 wasused to overexpress StarD1 in primary rat hepatocytes in order toincrease bile acid synthesis. In this study we present evidence forpreviously unappreciated sulfated oxysterols in the nucleus of cellsinfected with the StarD1 adenovirus. The chemical structure of the mostabundant nuclear oxysterol was characterized by HPLC, Triple QuadrupoleLC/MS/MS, enzymatic digestion, and TLC analysis and was identified as5-cholesten-3β, 25-diol 3-sulphate. We also provide evidence for apotential function(s) of this newly identified nuclear oxysterol incholesterol catabolism.

ABBREVIATIONS

The abbreviations used are: CYP8B1, sterol 12α-hydroxylase; CYP27A1,cholesterol 27-hydroxylase; CMV, cytomegalovirus; CYP7A1, cytochromeP450 7α-hydroxylase; FXR, famesil X receptor; SRE, sterol regulatoryelement; SREBP, sterol regulatory binding protein; HMG-CoA,3-hydroxy-3-methylglutaryl-coenzyme A; Q-RT-PCR, quantitative reversetranscription PCR.

Experimental Procedures Materials

Cell culture reagents and supplies were purchased from GIBCO BRL (GrandIsland, N.Y.). [¹⁴C]Cholesterol and [³H] 25-Hydroxycholesterol werepurchased from New England Nuclear (Boston, Mass.).[¹⁴C]27-Hydroxycholesterol was prepared as previously described (13).Cyclodextrin was purchased from Cyclodextrin Technologies DevelopmentInc. (Gainsville, Fla.). Silica gel thin-layer chromatography plates(LK6 D) were from Whatman (Clifton, N.J.). Silica gel 1B TLC sheets werepurchased from VWR (Bridgeport, N.J.). All other reagents were fromSigma Chemical Co (St. Louis, Mo.), unless otherwise indicated.

Adenovirus Preparation and Propagation

The adenovirus construct used in this study was obtained through theMassey Cancer Center Shared Resource Facility of the VirginiaCommonwealth University as previously described (14).

RNA Preparation and Quantification

RNA was isolated and CYP7A1 was quantified using Northern blot assays(20 μg of total RNA) as previously described (15).

Culture and Subcellular Fractionation of Primary Rat Hepatocytes andLipid Fractionation

Primary rat hepatocyte cultures, prepared as previously described (16),were plated on 150 mm tissue culture dishes (˜2.5×10⁷ cells) inWilliams' E medium containing dexamethasone (0.1 μM). Cells weremaintained in the absence of thyroid hormone. Twenty-four hrs afterplating, culture medium was removed, and 2.5 ml of fresh medium wasadded. Cells were then infected with recombinant adenovirus encodingeither the StarD1 (Ad-CMV-StarD1) or the CYP7A1 (Ad-CMV-CYP7A1) cDNAs infront of the human cytomegalovirus promoter (CMV) or no cDNA, as acontrol virus. The viruses were allowed to incubate for at least 2 hrsin minimal culture medium with gentle shaking of the plates every 15 minAfter 2 hrs of infection, unbound virus was removed, replaced with 20 mlof fresh medium, and 2.5 μCi of [¹⁴C]cholesterol was added. After 48hrs, cells were then harvested and processed for nuclei isolation asdescribed (17) with minor modification (FIG. 1). Briefly, cells weredisrupted by Dounce homogenization in buffer A (10 mM HEPES-KOH at pH7.6, 1.5 mM MgCl₂, 10 mM KCl, 0.5 mM dithiothreitol, 1 mM sodium EDTA, 1mM EGTA) and spun at 1,000×g for 10 min. The nuclear pellet was furtherfractionated by resuspension in 2.5 ml of a 1:1 mixture of buffer A andbuffer B (2.4M Sucrose, 15 mM KCl, 2 mM sodium EDTA, 0.15 mM spermine,0.15 mM spermidine, 0.5 mM dithiothreitol) and centrifuged at 100,000×gfor 1 hr at 4° C. through a 1 ml cushion of 3:7 mixture of buffer A andB. The washed nuclear pellet was resuspended in buffer A containing 0.5%(v/v) Nonidet P-40 and centrifuged at 1000×g for 10 min at 4° C. Thesupernatant is designated as nuclear attached membrane (fraction C) andthe pellets as purified nuclei (fraction D).

The purified nuclei (Fraction D) were resuspended and digested by 2mg/ml of DNase I in 50 mM of acetic buffer, pH 5.0, 10 mM MgCl₂ at 37°C. for 2 hrs. After centrifugation at 10,000×g for 20 min, thesupernatant was designed as inner nuclear membrane (fraction E). Thepellets were further digested by 2 mg/ml of proteinase K in phosphatebuffered saline solution (PBS) at 50° C. for 16 hrs and the solution wasdesigned as nuclear protease digests (fraction F). Total lipids in eachfraction were extracted by adding 3.3 volumes of chloroform:methanol(1:1) and separated into two phases, methanol/water and chloroformphases as previously described (18). The counts of [¹⁴C]cholesterolderivatives in the methanol/water and chloroform phases were measured byliquid scintillation counting (LC 60001C, Beckman, Fullerton, Calif.).

TLC and HPLC Analysis of Cholesterol Derivatives

The [¹⁴C]cholesterol/cholesterol derivatives in chloroform andmethanol/water phases were examined by thin layer chromatograph (TLC)(E. Merck, Darmstadt, Germany) using different developing solventsystems: toluene:ethyl acetate (2:3, v/v) for the[¹⁴C]cholesterol/oxysterols in chloroform phase, and ethylacetate:cyclohexane:acetic acid (92:28:12; v/v/v) for those inmethanol/water phases. The [¹⁴C]cholesterol/cholesterol derivatives werevisualized in Phosphorimager using Fuji imaging plates (FujifilmBAS-1800II, Fuji Photo Film Co., LTD, Japan).

Total [¹⁴C]-cholesterol derivatives in chloroform phase were analyzed byHPLC on an Ultrasphere Silica column (5μ×4.6 mm×25 cm; Backman, USA)using HP Series 1100 solvent delivery system (Hewlett Packard, Japan) at1.3 ml/min flow rate. The column was equilibrated and run in a solvent,Hexane:Isopropanol:Glacial Acetic Acid (965:25:10, v/v/v), as the mobilephase. The effluents were collected every 0.5 min (0.4 ml per fraction)except as indicated. The counts of [¹⁴C]cholesterol/cholesterolderivatives were determined by Scintillation Counter. The column wascalibrated with cholesterol, [³H]25-hydroxycholesterol, and[¹⁴C]27-hydroxycholesterol.

Total [¹⁴C]-cholesterol derivatives found in methanol/water phases wereanalyzed on an Ultrasphere PTH C-18 column (5μ×4.6 mm×25 cm; Backman,USA) at 0.8 ml/min flow rate. The column was equilibrated and run in 20mM KH₂PO₄, pH 4.2:acetonitrile:methanol (1:3:6, v/v/v) as the mobilephase. The effluents were monitored at 195 nm and collected every 0.5min (0.4 ml per fraction) except as indicated. The column was calibratedwith tauroursodeoxycholic acid, glycoursodeoxycholic acid, taurocholicacid, glycocholic acid, taurochenodeoxycholic acid, taurodeoxycholicacid, and progesterone. Sulfatase treatment of purified nuclear[¹⁴C]cholesterol derivatives:

The purified nuclear [¹⁴C]cholesterol derivatives were digested with 2mg/ml of sulfatase (EC 3.1.6.1) (Sigma, St Louis, Mo.) in 50 mM ofacetic buffer, pH 5.0 by incubation at 37° C. for 4 hrs. The productswere extracted into chloroform phase from methanol/water phase by adding3.3 volume of methanol:chloroform (1:1, v/v) to reaction solution.[¹⁴C]Cholesterol derivatives in both chloroform and methanol/waterphases were then analyzed by TLC and HPLC as stated above.

Reverse Phase Liquid Chromatography/Mass Spectrometry/Mass Spectrometry(LC/MS/MS) Analysis of Nuclear Cholesterol Derivatives:

Reverse phase liquid chromatographic separation was performed on an HPseries 1100 system (Agilent Technologies, Palo Alto, Calif.) and CTCHTS-PAL autosampler (Leap Technologies, Carrboro, N.C.). Separation wascarried on a ThermoKeystone Aquasil column (5 μm, 2.1 mm×100 mm). Themobile phase consisted of (A), 0.1% formic acid in water, and (B), 0.1%formic acid in acetonitrile. The 20 min gradient was as follows: 0-10.0min, 10%-95% B linear; 10.0-15.0 min, 95% B; 15.0-15.1 min, 95%-10% Blinear; 15.1-20.0 min, 10% B. The mass detector was an API 4000 (MDSSciex, Toronto, Canada)

The elution stream (0.3 ml/min) from the HPLC apparatus was introducedinto a MDS Sciex API 4000 Triple Quadrapole Mass Spectrometer with aTurbo IonSpray ionization (ESI) source for the analyses. The massspectrometer was operated in negative ion modes and data were acquiredusing both full scan mode as well as the product ion mode for MS/MS.

The sample was reconstituted into methanol:water (20:80, v/v). Thesolution containing the fraction of 11.50 min peak was infused into theLC/MS/MS system to optimize ESI-MS-MS parameters. The optimizedparameters for Q1 full scan under the negative mode were: CUR:10; GS1:40; GS2:40; TEM:400; IS:−4500; DP:−150; EP:−10. The optimized parametersfor the product scan of 481 under the negative mode were: CUR:10;GS1:40; GS2:40; TEM:400; IS:−4500; CAD:5; DP:−150; EP:−10; CE:50;CXP:−15.

Bile Acid Biosynthesis and Analysis:

Bile acid synthetic rates were determined by the addition of 2.5 μCi of[¹⁴C]cholesterol to each P150 mm plate of confluent primary rathepatocyte cultures (˜2.5×10⁷ cells) 24 hrs after plating. Media andcells were harvested 48 hrs after viral infection. Conversion of[¹⁴C]cholesterol into [¹⁴C]-methanol-water soluble products wasdetermined by scintillation counting after extraction withchloroform-methanol (2:1, vol/vol) of cells and of culture media. Ratesof bile acid biosynthesis following recombinant adenovirus infectionwere calculated as the ratio of [¹⁴C]-methanol-water soluble counts tothe sum of chloroform plus methanol-water counts. Individual bile acidswere identified by HPLC analysis as described above.

Time Course of Bile Acid Synthesis:

Time points for conversion of [¹⁴C]-cholesterol to [¹⁴C]-bile acids werecarried out using P150 mm tissue culture dishes Aliquots (1/100) ofmedia were collected in duplicate in a microfuge tube and kept frozenuntil analysis. A mini Folch extraction was carried out by adding 3volume of methanol:chloroform (1:1) to the culture medium. The tubeswere vigorously vortexed and centrifuged at 16,000 g for 6 min. Thephases were collected separately and counted.

Statistics:

Data are reported as mean±standard error. Where indicated, data weresubjected to t-test analysis and determined to be significantlydifferent if P<0.05.

Results A Novel Nuclear Oxysterol is Generated in Mitochondria andTranslocated to the Nucleus in Primary Rat Hepatocytes Upon StarD1Overexpression.

Primary rat hepatocytes were infected with an adenovirus encoding eitherStarD1 or CYP7A1 as explained in Experimental Procedures. Forty-eighthrs after infection, bile acid synthesis increased 4-fold and 7-foldrespectively, as we previously reported (18). Cells infected withcontrol virus have similar levels of bile acid synthesis as uninfectedcells. To test whether overexpression of StarD1 affects the expressionof LXR targeting genes following generation of the nuclear oxysterol,the regulation of CYP7A1 expression was investigated following StarD1overexpression. CYP7A1 mRNA levels increased by 6-fold (n=3) at day 3following StarD1 overexpression (FIG. 2).

The subcellular distribution of [¹⁴C]-cholesterol derivatives wasmonitored by adding exogenous [¹⁴C]cholesterol to the hepatocyte culture2 hrs after infection and is summarized in Table 1. Approximately 50% ofthe total counts of [¹⁴C]cholesterol/cholesterol derivatives were foundin nuclear-related fractions. The other 50% were located in othersubcellular organelles including cytosol, plasma membranes, lysosomes,and mitochondria (Fractions A and B). Only a small number of counts weredetected in the inner membrane fraction (Fraction E). Interestingly, thetotal extractable [¹⁴C]cholesterol/cholesterol derivatives in nucleardebris from StarD1-infected cells was significantly (25%) higher thanthose of CYP7A1-infected or control nuclear extracts (Fraction F). TLCanalysis of the chloroform-extractable cholesterol derivatives found inthe nuclear fraction (Fraction D) in StarD1-infected hepatocytes showedfour bands, which migrated like cholesterol ester, cholesterol,25-hydroxycholesterol, and 27-hydroxycholesterol respectively (FIG. 3).Only cholesterol ester, cholesterol, and 27-OH cholesterol were detectedin control-infected cells, with cholesterol ester and cholesterol inCYP7A1-infected cells. These results suggested that StarD1overexpression increased translocation of cholesterol derivatives,25-hydroxycholesterol, to the nucleus. The presence of25-hydroxycholesterol in the nucleus following StarD1 overexpression hasbeen confirmed by mass spectrometry/mass spectrometry (MS/MS) analysis(data not shown). 25-Hydroxycholesterol is a minor oxysterol that may beformed in different types of tissues by a specific enzyme that may notbelong to the cytochrome P-450 family (19). Interestingly,cholesterol-derivatives in the methanol/water phase extracted from thenuclear debris were dramatically increased in the StarD1-overexpressingcells compared with control and CYP7A1-overexpressing cells (FIG. 4).

TABLE 1 Distribution of [¹⁴C] cholesterol derivatives in primary rathepatocytes following StarD1 or CYP7A1 overexpression*. SubcellularNon-nuclear Nuclear Fraction A B C E F Control 43 ± 2  23 ± 5 47 ± 3 1.0± 0.2  7 ± 3 StarD1 38 ± 8 3.1 ± 2 36 ± 6 1.7 ± 0.1 10 ± 2 CYP7A1 43 ± 32.9 ± 4 49 ± 8 1.1 ± 0.2  8 ± 2 *[¹⁴C] Cholesterol was added at 2 hrsafter adenovirus infection and cells were harvested 48 hrs later.Subcellular fractions were prepared as described under ″ExperimentalProcedures″. An aliquot from each fraction was taken for liquidscintillation counting. Values represent the mean of three experiments±SD of the percentage of radioactivity found in each fraction withrespect to the total radioactivity found in all fractions.

We then proceeded to analyze the composition of the cholesterolderivatives in the different subcellular fractions of StarD1overexpressing hepatocytes. We first performed HPLC analysis of[¹⁴C]cholesterol derivatives in the methanol/water phases from thenuclear and non-nuclear fractions. FIG. 5 showed the 195 nm profiles,which nonspecifically detected double bonds containing molecules, andthe specific radioactivity incorporated in each fraction derived fromthe [¹⁴C]cholesterol that had been added to the cultures afterinfection. The 195 nm profiles were relatively similar in the nuclearfraction (Fraction D) from cells infected either with the StarD1,CYP7A1, or null recombinant viruses, except for an extra peak withretention time of 11.51 min, which was located between glycocholic acid(10.75 min) and taurochenodeoxycholic acid (12.50 min) (FIG. 5A). This11.50 min peak was the only fraction that contained ¹⁴C-labelledcholesterol derivatives and was detectable only in StarD1 overexpressingcells (FIG. 5B). The non-nuclear fraction (Fraction A) from the StarD1overexpressing cells contained two major [¹⁴C]cholesterol derivativepeaks with retention times at 5 and 11.50 min. The 11.50 min peak wasnot detected in cells infected with either the control or the CYP7A1viruses (FIG. 5C).

Further analysis of the cholesterol derivatives in the methanol/waterphases extracted from mitochondrial, nuclear fractions, and culturemedia following overexpression of StarD1 protein showed that two[¹⁴C]cholesterol derivatives with retention times at 5.00 min and 11.5min were found in mitochondria (FIGS. 6A and D). Furthermore, the 11.5min peak was present only in mitochondria and nuclei but not in themedia (FIGS. 6B and E) suggesting that a cholesterol-derived moleculeshould be generated in mitochondria and translocated to the nucleus. Thepeak with retention time at 5.00 min was found only in mitochondria andculture media, but not in the nucleus (FIGS. 6C and F) suggesting thatthe molecule presented in that peak was secreted from the cells.Therefore, the cholesterol derivative with a retention time of 11.50 minin the HPLC system, which is generated in the mitochondria andtranslocates to the nucleus, is defined as a nuclear oxysterol, and theother as a secretory oxysterol.

To characterize the chemical structure of the nuclear oxysterol, weproceeded to purify it by HPLC using C18 and silica columns, and analyzeit by enzymatic digestion, TLC, and HPLC analysis. First, to detect thepotential presence of sulfate group(s) in the nuclear oxysterolmolecule, the purified nuclear oxysterol was digested with sulfatase.The products were extracted with methanol/chloroform, separated by Folchpartitioning, and characterized by HPLC and TLC analysis. Followingsulfatase treatment, the nuclear oxysterol products were extracted intothe chloroform phase from the methanol/water phase and show the sameretention time as 25-hydroxycholesterol, but not 27-, no24-hydroxycholesterol in our HPLC system (FIGS. 7A-C), and the samerelative mobility as 25-hydroxycholesterol on the TLC plate (FIG. 7D),suggesting that the nuclear oxysterol derivative is a sulfated25-hydroxycholesterol.

To confirm the chemical structure of the nuclear oxysterol derivative,the purified nuclear sulfated oxysterol was further analyzed by LC/MS/MSMass Spectrometry. After we performed LC/MS (Q1 full scan mode, 350 to550 atomic mass units, amu) under negative ionization mode, a prominentpeak was observed at a retention time of 10.75 min in a selected ionchromatogram of mass ion at m/z=481 as shown in FIG. 8A. The Q1 fullscan spectrum showed the peak at 10.75 min mainly contained moleculeions, m/z 480.1 and 481.5 (FIG. 8B). Further analysis showed themolecule ion, m/z 480.1 did not contain a sulfate group on hydroxylgroup (m/z 97) (data not shown). However, the molecule ion, m/z 481.5,corresponds to sulfate group 97 and cholesterol (MW 386). This moleculeion was further analyzed by LC/MS/MS (product scan of m/z 481) undernegative ionization mode (FIG. 8C). The characteristic fragment ionswere observed at m/z=80(a), 97(b), 107, 123(c), 288, 465, and 59(d) inthe product scan spectrum of m/z 481 (FIG. 8C). These observed fragmentions indicate that the nuclear oxysterol is a sulfated oxysterol with asulfate group on 3-OH position (20) and a hydroxyl group on side chain,m/z 59, (molecular mass 482=80 (sulfate)+16 (0)+386 (cholesterol).Combined with data from HPLC, enzymatic digestion, and TLC analysis, thenuclear oxysterol derivative can be characterized as 5-cholesten-3β,25-diol 3-sulphate (25-hydroxycholesterol 3-sulfate) as shown in (FIG.8C).

Nuclear Oxysterol derivatives from Primary Rat HepatocytesOverexpressing StarD1 Increase Cholesterol Uptake and Bile AcidSynthesis.

To examine the function of the newly characterized nuclear oxysterolderivative in cholesterol homeostasis, the effects of the purifiedoxysterol on cholesterol uptake and bile acid synthesis were determinedby measurement of cholesterol and conversion of [¹⁴C]cholesterol intomethanol/water-extractable [¹⁴C]products in primary rat hepatocytes. Therate of cholesterol uptake was slightly higher in cells treated withnuclear extracts extracted from StarD1 overexpressing cells than thosein hepatocytes treated with nuclear extracts isolated from CYP7A1overexpressing cells or from control cells (data not shown). Similarly,rates of bile acid synthesis were significantly higher in hepatocytestreated with nuclear extracts from StarD1 overexpressing cells, andincreased by 4-fold at 24 hrs after addition of the nuclear extracts. Incontrast, nuclear extracts from CYP7A1 overexpressing cells did notsignificantly change bile acid synthetic rates compared with controlnuclear extracts (FIG. 9A). To further confirm the role of the nuclearoxysterol derivatives in bile acid biosynthesis, the purified oxysterolderivative was dissolved in nuclear extracts (methanol/water phase) fromcells infected with the control virus and added to primary rathepatocytes. The results were very similar to those in cells directlytreated with the nuclear extracts from the StarD1 overexpressing cellsand in a time- and concentration-dependent manner (FIG. 9B), providingevidence that the nuclear oxysterol is a potent regulator of bile acidsynthesis.

Discussion

Evidence for the first time to identify a novel nuclear regulatoryoxysterol derivative, which is generated in mitochondria, translocatedto the nucleus, and upregulates bile acid synthesis, gives rise to a newhypothesis that a new regulatory transduction pathway may play animportant role in maintenance of intracellular cholesterol homeostasis.

The present results suggested that mitochondrial cholesterol transportproteins, such as StarD1, could serve as sensors of intracellularcholesterol levels. When cholesterol levels increase, StarD1 proteinsdeliver cholesterol into mitochondria where it is metabolized to 25-OHcholesterol 3-sulfate (the nuclear sulfated oxysterol). The generatednuclear oxysterol derivative is then translocated into the nucleusprobably by binding and activating the nuclear oxysterol receptor(s).Without being bound by theory, it appears plausible that the nuclearsulfated oxysterol-nuclear receptor(s) complex may enter into the nucleiand regulate gene expression involved in cholesterol metabolism. Apossible mechanism is proposed in FIG. 11.

25-Hydroxycholsterol 3-sulfate May be a Potential Authentic Ligand ofNuclear Sterol Receptor(s).

In the presence of StarD1 protein, cholesterol enters the mitochondriawhere it is oxidized to 25-hydroxycholesterol and is then sulfated tothe nuclear sulfated oxysterol. The present results showed that both25-hydroxycholesterol and 25-hydroxycholesterol 3-sulfate entered thenucleus. To date, 25-hydroxycholesterol has been believed to be the mostpotent regulator of gene transcription (21-23). To identify which oneexhibits a more potent regulatory function, 25-hydroxycholesterol and25-hydroxycholesterol 3-sulfate in nuclear extracts were separated byFolch partitioning: 25-hydroxycholesterol partitions to the chloroformphase and 25-hydroxycholesterol 3-sulfate partitions to themethanol/water phase. Interestingly, addition of the chloroform phaseextracts containing 25-hydroxycholesterol to cells slightly increasedthe rates of bile acid synthesis (data not shown). However, the additionof methanol phase extracts dramatically increased bile acid synthesis(FIG. 9). Moreover, 25-hydroxycholesterol 3-sulfate is a water-solublecompound. It is thus reasonable to propose that 25-hydroxycholesterol3-sulfate serves as a potent nuclear sterol regulators in vivo.

25-Hydroxylases (25-OHLase) and Hydroxycholesterol Sulfotransferase 2(HST2) May be Involved in the Generation of the Potent Nuclear SulfatedOxysterol.

StarD1 delivers cholesterol into the mitochondria and generates a novelnuclear sulfated oxysterol, 25-hydroxycholesterol 3-sulphate. This datasuggests that a new pathway of cholesterol metabolism is responsible forgenerating this nuclear oxysterol. Our present report shows the presenceof 25-hydroxycholesterol 3-sulfate in both the mitochondria and thenucleus but not in culture media, suggesting that 25-hydroxycholesterol3-sulfate is generated in mitochondria and translocates exclusively tothe nucleus. To biosynthesize this potent nuclear sterol regulator, tworeactions should be involved: 25-hydroxylation and 3β-sulfation ofcholesterol. It is not clear at this time whether 25-hydroxylation iscatalyzed by CYP27A1 or 25-hydroxylase because 25-hydroxylase has notyet been identified in hepatocytes or in mitochondria, although it hasbeen cloned (19). Since we did not see any cholesterol-3β-sulfate in theHPLC elution profile, we believe the first step is likely to be25-hydroxylation of cholesterol catalyzed by 25-hydroxylase to form25-hydroxycholesterol. Subsequently, sulfation of 25-hydroxycholesterolat the 3β position, is catalyzed by hydroxycholesterol sulfotransferasesHST2(a, b), enzymes which have recently been cloned and identified(24;25). In addition, 25-hydroxycholesterol 3-sulfate may beglucuronidated for further catabolism and secretion via the bile as24-hydroxycholesterol 3-sulfate (26). However, at the present time it isnot clear that the [¹⁴C]-cholesterol derivative with a retention time of5 min is the glucuronidation product of 25-hydroxycholesterol 3-sulfate(FIGS. 5 and 6).

Steroid sulfate conjugates may play an important role in the maintenanceof cholesterol homeostasis. An interesting development in recent yearshas been the realization that steroid sulfoconjugates play importantroles in well-characterized biological effects, such as serving aspotent neuroexcitatory agents, which are distinct from the well-knownrole of unconjugated steroids as ligands for nuclear receptors toregulate gene expression (27). Several sulfated sterols have beenreported to be widely distributed in steroidogenesis tissues (26) and tocirculate in plasma at concentrations ranging from 328-924 μg/100 ml,with a blood production rate of 35-163 mg/day (28). A similar sulfatedoxysterol derivative, 24-hydroxycholeterol 3-sulfate 24-glucuronide(based on MS/MS analysis) was reported to be in the serum and urine ofchildren with severe cholestatic liver diseases (26). The 3-sulfate of24-hydroxycholesterol is the major hydroxycholesterol sulfate found inmeconium and infant feces (29;30), and is most likely excreted via bile.Bile excretion of this oxysterol would be impaired in cholestasis,leading to its increased concentration of 24-hydroxycholesterol3-sulfate in hepatocytes and conceivably leading to its glucuronidation.

However, where the compound came from and what function this oxysterolplays in the cholestatic liver is still a mystery. Although high levelsof the double conjugate of 24-hydroxycholesterol are an indicator ofgrave liver disease, and can be used as a criterion for recommendingliver transplantation, the physiological role and metabolism of thiscompound have never been identified. Sulfated sterols have beenimplicated in a wide variety of biological processes, e.g. regulation ofcholesterol synthesis, sperm capacitation, thrombin and plasminactivities, and activation of protein kinase C isozymes (24).Furthermore, sulfated sterols can serve as a substrate for adrenal andovarian steroidogenesis (31;32). Sulfated sterols play an important, butunclear, role in the normal development and physiology of skin, where anepidermal sterol sulfate cycle has been described (24). The presentresults show that the nuclear extract containing the sulfated oxysteroland the purified nuclear sulfated oxysterol dramatically increased therates of bile acid synthesis, strongly suggesting that the nuclearsulfated oxysterol may play an important role in cholesterol metabolism.

Activation of LXRs by oxysterols is believed to be responsible forregulation of the metabolism of several important lipids, includingcholesterol and bile acids (33). The identification of an LXR responseelement in the promoter of the rat cholesterol CYP7A1 suggested thatLXRs play an important role in the regulation of cholesterol homeostasis(34;35). LXRα-deficient mice (LXRα−/−) dysregulate the CYP7A1 gene andseveral other important lipid-associated genes (3). Studies utilizingthese animals confirmed the essential function of LXRα as a major sensorof dietary cholesterol and an activator of the bile acid syntheticpathway in mice. The finding of authentic oxysterol ligands for LXRs isone of the most important investigative methods for developing newtherapeutic methods for the prevention and treatment of hyperlipidemiaand atherosclerosis. To date, there is still only indirect evidence ofthe important role of oxysterols, such as 24-, 25-, or27-hydroxycholesterol, as authentic ligands in the normal regulation ofcholesterol homeostasis. Soluble and nuclear oxysterol-binding proteinswith a high affinity for oxysterols exist, but the physiological ligandsfor these proteins have not yet been defined with certainty (36). Thepresent report, with evidence showing that a potent regulatory nuclearsulfated oxysterol that is generated in the mitochondria translocatesinto the nucleus, provides a new clue regarding the role of oxysterol(s)in the regulation of intracellular cholesterol homeostasis. It isreasonable to hypothesize that the regulatory nuclear sulfated oxysterolgenerated in mitochondria translocates into nucleus, activates nuclearoxysterol receptor(s), and up-regulates bile acid synthesis. The nuclearsulfated oxysterol serves as a ligand of nuclear sterol receptor(s).

REFERENCES FOR EXAMPLE 1

-   1. Russell, D. W. (2003) Annu. Rev. Biochem. 72, 137-174-   2. Hylemon, P. B., Gurley, E. C., Stravitz, R. T., Litz, J. S.,    Pandak, W. M., Chiang, J. Y., and Vlahcevic, Z. R. (1992) J. Biol.    Chem. 267, 16866-16871-   3. Chiang, J. Y., Kimmel, R., and Stroup, D. (2001) Gene 262,    257-265-   4. Saucier, S. E., Kandutsch, A. A., Taylor, F. R., Spencer, T. A.,    Phirwa, S., and Gayen, A. K. (1985) J. Biol. Chem. 260, 14571-14579-   5. Schroepfer, G. J., Jr. (2000) Physiol Rev. 80, 361-554-   6. Szanto, A., Benko, S., Szatmari, I., Balint, B. L., Furtos, I.,    Ruhl, R., Molnar, S., Csiba, L., Garuti, R., Calandra, S., Larsson,    H., Diczfalusy, U., and Nagy, L. (2004) Mol. Cell Biol. 24,    8154-8166-   7. Dubrac, S., Lear, S. R., Ananthanarayanan, M., Balasubramaniyan,    N., Bollineni, J., Shefer, S., Hyogo, H., Cohen, D. E., Blanche, P.    J., Krauss, R. M., Batta, A. K., Salen, G., Suchy, F. J., Maeda, N.,    and Erickson, S. K. (2005) J. Lipid Res. 46, 76-85-   8. Fu, X., Menke, J. G., Chen, Y., Zhou, G., Macnaul, K. L.,    Wright, S. D., Sparrow, C. P., and Lund, E. G. (2001) J. Biol. Chem.    276, 38378-38387-   9. Bjorkhem, I. (2002) J. Clin. Invest 110, 725-730-   10. Pandak, W. M., Ren, S., Marques, D., Hall, E., Redford, K.,    Mallonee, D., Bohdan, P., Heuman, D., Gil, G., and    Hylemon, P. (2002) J. Biol. Chem. 277, 48158-48164-   11. Ren, S., Hylemon, P. B., Marques, D., Gurley, E., Bodhan, P.,    Hall, E., Redford, K., Gil, G., and Pandak, W. M. (2004) Hepatology    40, 910-917-   12. Hall, E. A., Ren, S., Hylemon, P. B., Rodriguez-Agudo, D.,    Redford, K., Marques, D., Kang, D., Gil, G., and    Pandak, W. M. (2005) Biochim Biophys. Acta 1733, 111-119-   13. Rodriguez-Agudo, D., Ren, S., Hylemon, P. B., Redford, K.,    Natarajan, R., Del Castillo, A., Gil, G., and    Pandak, W. M. (2005) J. Lipid Res. 46, 1615-1623-   14. Pandak, W. M., Schwarz, C., Hylemon, P. B., Mallonee, D.,    Valerie, K., Heuman, D. M., Fisher, R. A., Redford, K., and    Vlahcevic, Z. R. (2001) Am. J. Physiol Gastrointest. Liver Physiol    281, G878-G889-   15. Ren, S., Marques, D., Redford, K., Hylemon, P. B., Gil, G.,    Vlahcevic, Z. R., and Pandak, W. M. (2003) Metabolism 52, 636-642-   16. Pandak, W. M., Bohdan, P., Franklund, C., Mallonee, D. H.,    Eggertsen, G., Bjorkhem, I., Gil, G., Vlahcevic, Z. R., and    Hylemon, P. B. (2001) Gastroenterology 120, 1801-1809-   17. Dignam, J. D., Lebovitz, R. M., and Roeder, R. G. (1983) Nucleic    Acids Res. 11, 1475-1489-   18. Ren, S., Hylemon, P., Marques, D., Hall, E., Redford, K., Gil,    G., and Pandak, W. M. (2004) J. Lipid Res. 45, 2123-2131-   19. Lund, E. G., Kerr, T. A., Sakai, J., Li, W. P., and    Russell, D. W. (1998) J. Biol. Chem. 273, 34316-34327-   20. Lemonde, H. A., Johnson, A. W., and Clayton, P. T. (1999) Rapid    Commun. Mass Spectrom. 13, 1159-1164-   21. Kandutsch, A. A., Taylor, F. R., and Shown, E. P. (1984) J.    Biol. Chem. 259, 12388-12397-   22. Lehmann, J. M., Kliewer, S. A., Moore, L. B., Smith-Oliver, T.    A., Oliver, B. B., Su, J. L., Sundseth, S. S., Winegar, D. A.,    Blanchard, D. E., Spencer, T. A., and Willson, T. M. (1997) J. Biol.    Chem. 272, 3137-3140-   23. Peet, D. J., Turley, S. D., Ma, W., Janowski, B. A.,    Lobaccaro, J. M., Hammer, R. E., and Mangelsdorf, D. J. (1998) Cell    93, 693-704-   24. Javitt, N. B., Lee, Y. C., Shimizu, C., Fuda, H., and    Strott, C. A. (2001) Endocrinology 142, 2978-2984-   25. Her, C., Wood, T. C., Eichler, E. E., Mohrenweiser, H. W.,    Ramagli, L. S., Siciliano, M. J., and Weinshilboum, R. M. (1998)    Genomics 53, 284-295-   26. Meng, L. J., Griffiths, W. J., Nazer, H., Yang, Y., and    Sjovall, J. (1997) J. Lipid Res. 38, 926-934-   27. Paul, S. M. and Purdy, R. H. (1992) FASEB J. 6, 2311-2322-   28. Gurpide, E., Roberts, K. D., Welch, M. T., Bandy, L., and    Lieberman, S. (1966) Biochemistry 5, 3352-3362-   29. Montelius, J., Gustafsson, J. A., Ingelman-Sundberg, M., and    Rydstrom, J. (1977) Biochim Biophys. Acta 488, 502-511-   30. Gustafsson, J. A. and Sjovall, J. (1969) Eur. J. Biochem. 8,    467-472-   31. Korte, K., Hemsell, P. G., and Mason, J. I. (1982) J. Clin.    Endocrinol. Metab 55, 671-675-   32. Tuckey, R. C. (1990) J. Steroid Biochem. Mol. Biol. 37, 121-127-   33. Komuves, L. G., Schmuth, M., Fowler, A. J., Elias, P. M.,    Hanley, K., Man, M. Q., Moser, A. H., Lobaccaro, J. M., Williams, M.    L., Mangelsdorf, D. J., and Feingold, K. R. (2002) J. Invest    Dermatol. 118, 25-34-   34. Edwards, P. A., Kennedy, M. A., and Mak, P. A. (2002) Vascul.    Pharmacol. 38, 249-256-   35. Peet, D. J., Janowski, B. A., and Mangelsdorf, D. J. (1998)    Curr. Opin. Genet. Dev. 8, 571-575-   36. Bjorkhem, I. and Diczfalusy, U. (2002) Arterioscler. Thromb.    Vasc. Biol. 22, 734-742

Example 2 Demonstration of Up-Regulation of LXR Targeting GeneExpression

Preliminary experiments have shown that the purified nuclear oxysterolup-regulates bile acid synthesis. To further investigate the mechanismof this activity, the effect of purified 5-cholesten-3β, 25-diol3-sulphate on gene expression of LXR-targeted cholesterol transportproteins ABCA1, ABCG1, ABCG5, ABCG8, and LDLR was investigated. Purifiednuclear sulfated oxysterol was added to primary hepatocytes in culture,and mRNA levels of the transport proteins were quantitated by real timeRT-PCR. The primer sets and TagMan probes for detection of mRNA levelswere purchased from AB Applied Biosystem (Foster City, Calif.) and thereactions were performed on an MJ Research DNA Engine Opticoninstrument. The results are presented in FIG. 12 and show that theaddition of purified nuclear oxysterol to primary hepatocytes in cultureincreased expression of ABCA1 (2-fold) and ABCG5 (1.6-fold), and ABCG8(3 fold). The expression of ABCG1 is typically low in primaryhepatocytes and did not change compared with control cells. Thecorrelation of increased levels of the sulfated oxysterol in nuclei withincreased levels of LXR targeting gene expression suggests that thenuclear oxysterol receptor, LXR, is activated by exposure to thesulfated oxysterol. These data do not rule out the activation of othernuclear receptor(s) as well.

Example 3 Synthesis of 5-cholesten-3β, 25-diol 3-sulphate

FIG. 12A shows a schematic illustration of the synthesis of the novelsulfated oxysterol of the invention by addition of a sulfate group tothe 3β-position of 25-hydroxycholesterol. Synthesis was carried out asfollows: A mixture of 25-hydroxycholesterol (0.1 mmol) and sulfurtrioxide triethyl amine complex (0.12 mmol) in dry toluene was heated to60 degree for 24 hours under nitrogen, then cooled and the solvent wasevaporated under reduced pressure. The residue was purified by flashchromatography to afford the product as a white solid using the methodof described above. FIG. 12B shows the mass spectrophotometric analysisof the HPLC purified product, which suggests that the sulfate group hasbeen successfully added to 25-hydroxycholesterol.

FIGS. 12C and 12D show NMR data for the starting material,25-hydroxycholesterol, and product, respectively. As can be seen, theresonance of C3 in the molecule has been shifted from 3.35 ppm in theoriginal compound to 4.12 ppm in the product, suggesting that thedesired product, 3β-sulfated 25-hydroxycholesterol, has been formed.

Example 4 A Nuclear Oxysterol, 25HC3S, Decreases Cholesterol Synthesisvia Inhibition of SREBP-1 Activation in Hepatocytes Abstract

Recently, a novel oxysterol, 5-cholesten-3β, 25-diol 3-sulfate (25HC3S)was discovered, characterized, and identified in mitochondria and nucleiof primary hepatocytes following overexpression of the cholesteroltransport protein, StarD1. This oxysterol was also detected in humanliver nuclei. In the present study, 25HC3S was chemically synthesized.Addition of varying concentrations of 25HC3S to HepG2 cells markedlyinhibited cholesterol biosynthesis and significantly decreasedmicrosomal cholesterol. Real time RT-PCR and Western blot analysis showsthat 25HC3S strongly decreased HMG CoA reductase mRNA levels in HepG2cells and primary human hepatocytes. In comparison,25-hydroxycholesterol (25HC) inhibited HMG CoA reductase mRNA levels inHepG2 cells but not in primary human hepatocytes when the cells wereculture in serum-free media. Coincidentally, 25HC3S inhibited theactivation of steroid response element binding protein (SREBP-1) inabsence or presence of mevinolin and mevalonate, indicating thatcholesterol biosynthesis inhibition occurred through blocking SREBP-1activation, and subsequently the expression of HMG CoA reductase in thehuman hepatocytes. In conclusion, the presented findings indicate that25HC3S regulates intracellular cholesterol biosynthesis via inhibitingSREBPs activation in human hepatocytes.

Introduction

The “acidic” pathway of bile acid biosynthesis is initiated by themitochondrial enzyme sterol 27-hydroxylase (CYP27A1). Oxysterolintermediates of the “acidic” pathway such as 27-hydroxycholesterol(27HC) and 25-hydroxycholesterol (25HC) have been shown to be regulatorsof cholesterol homeostasis (1;2). These oxysterols represent regulatorymolecules for the expression of many other genes encoding enzymesinvolved in cholesterol biosynthesis and transport (3-5). In theory,increased CYP27A1 activity in peripheral tissues could bothdown-regulate cholesterol synthesis through generating regulatoryoxysterols and the steroid response element binding proteins (SREBPs)pathway, and enhance the cellular efflux of cholesterol, i.e. itselimination, via liver oxysterol receptor, LXR (6). However, therelationship between the CYP27A1 activity and intracellular cholesterolmetabolism is unknown.

Previous reports showed that overexpression of the steroidogenic acuteregulatory protein (StarD1), a protein which facilitates cholesteroltransport into mitochondria, dramatically increases cholesterolcatabolism to bile acids both in primary hepatocytes in culture and invivo (7;8). These findings suggest that cholesterol delivery to themitochondria, where the enzyme CYP27A1 is localized, is therate-determining step for bile acid synthesis via the “acidic” pathway.Subsequently, StarD1 was detected in hepatocytes (9). Overexpression ofStarD1 in vivo not only increases bile acid synthesis to the same levelas overexpression of CYP7A1, but also produces a similar composition ofbile acids in bile (8). As described in Examples 1-3, a novel oxysterol,5-cholesten-3β, 25-diol 3-sulfate (25HC3S) was found and characterizedin mitochondria and nuclei of primary hepatocytes followingoverexpression of StarD1. This oxysterol was also present in human livernuclei (10). The results suggested that the oxysterol is synthesized inthe mitochondria and tranlocated to the nuclei. Oxysteorls in nucleishould be able to play important roles in maintenance of intracellularcholesterol homeostasis. However, the function of this nuclearoxysterol, 25HC3S, remains unknown.

In the present study, we chemically synthesized 25HC3S and presentedevidence that this oxysterol strongly regulates HMG CoA reductase, a keyenzyme in cholesterol biosynthesis via SREBP regulatory system inhepatocytes.

Materials and Methods

Materials:

Cell culture reagents and supplies were purchased from GIBCO BRL (GrandIsland, N.Y.); [¹⁴C]Cholesterol and [³H]25-hydroxycholesterol ([³H]25HC)from New England Nuclear (Boston, Mass.). [¹⁴C]27-OH Cholesterol wasprepared as previously described (11). HepG2 cells were obtained fromAmerican Type Culture Collection (Rockville, Md.). Fetal bovine serumwas obtained from Bio Whittaker (Walkersville, Md.). Tissue cultureflasks were purchased from Costar Corp. (Cambridge, Mass.). The reagentsfor real time RT-PCR were from AB Applied Biosystem (Warrington WA1 4SR, UK). The chemicals used in this research were obtained from SigmaChemical Co. (St. Louis, Mo.) or Bio-Rad laboratories (Hercules, Calif.)unless otherwise specified. All solvents were obtained from Fisher (FairLawn, N.J.) unless otherwise indicated. The enhanced chemilluminescence(ECL) reagents were purchased from Amersham Biosciences (Piscataway,N.J.). The testosterone and 27HC were obtained from Research Plus Inc.(Bayonne, N.J.). LK6 20×20 cm thin layer chromatography (TLC) plateswere purchased from Whatman Inc. (Clifton, N.J.). Nylon membranes werepurchased from Micron Separation Inc. (Westborough, Mass.).

Chemical Synthesis of 5-cholesten-3/3, 25-diol 3-sulfate

A mixture of 25HC (402 mg, 1 mmol) and triethylamine-sulfur trioxidepyridine complex (160 mg, 1 mmol) in 5 ml of dry pyridine was stirred atroom temperature for 2 hrs. After the solvent was evaporated at reducedpressure, the products were purified by HPLC using silica gel column andsolvent of methylene chloride and methanol (5%) as mobile phase toafford the product as a white solid powder. The structure of the productwas characterized by mass spectrum (MS) and nuclear magnetic resonance(NMR) spectroscopy analysis.

Mass Spectral Analysis

The synthesized compound was analyzed by a MDS Sciex ABI 4000 TripleQuadrapole Mass Spectrometer (MDS Sciex, Toronto, Canada) with a TurboIonSpray ionization (ESI) source and the mass spectrometer was operatedin negative ion modes and data were acquired using full scan mode aspreviously described (10).

Proton Nuclear Magnetic Resonance Spectroscopy

Samples were prepared for ¹H NMR analysis as described previously (12).Briefly, each sample, 1 mg, was dissolved in 0.5 ml of D₂O andlyophilized to remove exchangeable protons. The residue was dissolved in0.5 ml of dimethyl sulfoxide-d6/D₂O (98:2, v/v). NMR spectra wereobtained on spectrometers operating at ¹H frequencies of 300 MHz.

Cell Culture

HepG2 cells were grown in MEM containing non-essential amino acids,0.03M NaHCO₃, 10% FBS, 1 mM L-glutamine, 1 mM sodium pyruvate and 1%Pen/Strep and incubated at 37° C. in 5% CO₂. When cells reached at ˜90%confluency, the oxysterols in DMSO or in ethanol (final concentration,0.1%) and/or [1-¹⁴C]acetate for cholesterol synthesis was added orotherwise as indicated. Microsomal and cytosol fractions were isolatedfrom broken cells as described previously (10).

Primary human hepatocytes were purchased from an NIH-approved facility(Liver Tissue Procurement Distribution System, Univ. of Minnesota).Cells were obtained from a random sampling of males and females 18-69 yrof age. Experiments were performed as cells became available tocorroborate findings in experiments conducted in HepG2 cells as previousdescribed (13).

Determination of Cholesterol Biosynthesis by TLC and HPLC

After incubation of HepG2 cells with media containing 25HC3S for 6 hrs,cultures in 25-cm2 flasks were given 3 ml of the same fresh mediumcontaining 5 μCi of the [1-¹⁴C]acetate. After 4 hr incubation at 37° C.,the media were removed and the cells were washed twice withphosphate-saline buffer (PBS), harvested with rubber police asdescribed, and collected in Eppendoff tubes. The cells were sedimentedby centrifugation and the pellets were washed three times byresuspension and sedimentation. The pellets were resuspended in 0.3 mlof PBS. The total lipids were extracted and separated by adding 3 volumeof chloroform:methanol (v/v, 1:1). [¹⁴C]Cholesterol and27-hydroxycholesterol was isolated into chloroform phase and separatedon TLC (tuluene:acetyl acetate, 2/3, v/v/). [1-¹⁴C]acetate derivativeswere visualized by Image Reader, Fujifilm BAS-1800 II.

HPLC Analysis of Cholesterol Derivatives

[1-¹⁴C]Acetate derivatives in the chloroform phase were analyzed by HPLCon an Ultrasphere Silica column (5μ×4.6 mm×25 cm; Backman, USA) using HPSeries 1100 solvent delivery system (Hewlett Packard) at 1.3 ml/min flowrate. The column was equilibrated and run in a solvent system ofhexane:isopropanol:glacial acetic acid (965:25:10, v/v/v), as the mobilephase. The effluents were collected every 0.5 min (0.65 ml per fraction)except as indicated. The counts in [¹⁴C]acetate derivatives weredetermined by Scintillation Counting. The column was calibrated with[¹⁴C]cholesterol, [³H]25HC, and [¹⁴C]27-hydroxycholesterol.

Determination of HMG CoA Reductase mRNA by Real-Time RT-PCR

Total RNA was isolated from HepG2 cells using SV Total RNA Isolation Kit(Promega). Two μg of total RNA was used for first-strand cDNA synthesisas recommended by manufacture (Invitrogen). Real-time PCR was performedusing SYBR Green on ABI 7500 Fast Real-Time PCR System (AppliedBiosystems). The final reaction mixture contained 5 ng of cDNA, 100 nMof each primer, 10 μl of 2×SYBR® Green PCR Master Mix (AppliedBiosystems), and RNase-free water to complete the reaction mixturevolume to 20 μl. All reactions were performed in triplicate. The PCR wascarried out for 40 cycles at 95° C. for 15 s and 60° C. for 1 min. Thefluorescence was read during the reaction, allowing a continuousmonitoring of the amount of PCR product. The data was normalized tointernal control-β-actin or GAPDH mRNA. The sequences of primers for HMGCoA reductase used in real-time PCR are ACCTTTCCAGAGCAAGCACATT (SEQ IDNO: 1) (Forward) and AGGACCTAAAATTGCCATTCCA (SEQ ID NO: 2) (Reverse).

Western Blot Analysis

Microsomal proteins (80 μg) were separated on a 7.5% SDS-polyacrylamidedenaturing gel according to the method of Laemmli (14). FollowingSDS-PAGE (Hoefer Vertical Slab Gel Unit; Heofer, San Francisco, Calif.),proteins were electrophoretically transferred overnight (4° C.) toImmobilon-P membranes using a Hoefer Trans Blot Electrophoretic Transfercell. The membranes were then blocked for 90 minutes (25° C.) inblocking buffer (PBS, pH 7.4, 0.1% Tween, 5% non-fat dry milk). Proteinswere then incubated for 90 minutes (25° C.) or overnight (4° C.) with arabbit polyclonal IgG (1:2500-1:20,000) against human SREBP-1, SREBP-2,or HMG CoA reductase. After washing, a secondary antibody (goatanti-rabbit IgG-Horse-radish peroxidase conjugate, 1:2500) was added tothe blocking solution (25° C., 90 minutes). Protein bands were detectedusing the Amersham ECL plus Kit.

Results

Chemical synthesis of the nuclear oxysterol, 5-cholesten-3β, 25-diol3-Sulfate To study the role that 25HC3 S may play in cellularcholesterol homeostasis, this oxysterol was chemically synthesized.Using a modified triethylamine-sulfur trioxide complex protocol asdescribed in Methods, the nuclear oxysterol was successfully synthesizedas outlined in FIG. 12A. MS analysis of the synthesized compound showsthe same molecular mass ion, m/z 481 as the “authentic” nuclearoxysterol (FIG. 12B). ¹H NMR analysis shows that the proton resonance atC3 with multiple small (1.5 Hz) splits near 3.65 ppm in the spectrum of25-OH cholesterol (starting material) (FIG. 12C) is shifted to 4.20 ppmin the product spectrum (FIG. 12D) as indicated, which confirms thatHSO₃ ⁻ group is added at the C3 position of 25-OH cholesterol. HPLCanalysis shows the same retention time of the synthesized oxysterol asthe authentic nuclear oxysterol (data not shown). Combined with theresults from the MS analysis, it is concluded that the synthesizedmolecule is 5-cholesten-3β, 25-diol 3-sulphate (25HC3S). The resultsalso confirmed the structure of the nuclear oxysterol as previouslyreported (10 and Examples 1-3).

25HC3S Inhibits Cholesterol Biosynthesis and Decreases CholesterolLevels in Microsomal Fractions

To examine the effects of 25HC3S on cholesterol biosynthesis, the ratesof cholesterol synthesis were determined by adding [1-¹⁴C]acetatefollowing the addition of varying concentration of 25HC3S to HepG2 cellsin culture. FIG. 13 summarizes the effects of 25HC3S on cholesterolbiosynthesis. After incubation of the cells in the media containing25HC3S for 6 hrs and [¹⁴C]acetate for additional 4 hrs, total lipidswere extracted and partitioned. Neutral lipids in the chloroform phasesincluding [¹⁴C]acetate derivatives were analyzed by TLC and HPLC, TLCanalysis shows that [¹⁴C]cholesterol ether, [¹⁴C]cholesterol, and[¹⁴C]25HC were synthesized but no detectable [¹⁴C]27HC (FIG. 13A). Free[¹⁴C]cholesterol was found to be significantly decreased followingaddition of 25HC3S while the other labeled sterols did not significantlychange. The decreases were concentration dependent (FIG. 13A). Thedecreased amounts of [¹⁴C]cholesterol bands on the TLC were confirmed byHPLC analysis as shown in FIG. 13B. The results from three experimentsare summarized in FIG. 13C.

To study the distribution of intracellular cholesterol following theaddition of 25HC3S, microsomal, nuclear, and cytosol fractions wereisolated and total lipids from each fraction were extracted withchloroform/methanol/water. The cholesterol concentration in eachfraction was determined by HPLC after cholesterol oxidase treatmentwhich converts sterols to 3-oxo-β4 derivatives. As shown in FIG. 14,25HC3S significantly decreased cholesterol levels in the microsomalfraction (left panels) as well as 25HC (right panels) but not in cytosoland nuclear fractions (data not shown). The decreases in cholesterolconcentration were dose dependent as indicated in FIG. 14. It was alsoobserved that no 25HC could be detected in the fractions from cellstreated with 25HC3S. In contrast, dose dependent levels of25-hydroxycholeterol could be detected in the fractions treated with25HC as shown in FIG. 14 (right panels). These results suggest that25HC3S directly inhibits cholesterol biosynthesis and decreasescholesterol levels in microsomal fractions and not through itsdegradation to be 25HC.

The Nuclear Oxysterol Inhibits Cholesterol Biosynthesis by DecreasingHMG CoA Reductase mRNA Levels

To investigate how 25HC3 S inhibits cholesterol biosynthesis, total mRNAwere isolated from HepG2 cells following incubation in 10% FBS freshmedia containing different concentrations of 25HC3S (FIG. 15A). The mRNAlevels of HMG CoA reductase were determined by real time RT-PCR. Asshown in FIG. 15A, there was concentration dependent decreases in HMGCoA reductase mRNA following the addition of 25HC3S to the cells inculture. The addition of 25HC3S to HepG2 cells also lead to a markeddecrease in the levels of HMG CoA reductase protein (FIGS. 15C and D).Western blot analysis shows that 50% of its protein level was decreasedin a concentration dependent following the addition of 25HC3S to culturemedia (FIGS. 15C and D).

To compare with 25HC, mRNA levels of HMG CoA reductase in 25HC or25HC3S-treated HepG2 cells were analyzed by real time RT-PCR. Both ofthe compounds can inhibit HMG CoA reductase in a similar fashion asshown in FIG. 16A. To further distinguish whether 25HC3S inhibits HMGCoA reductase expression via lipids uptake in the culture media, HepG2cells were incubated in media containing lipid-depleted serum for 2 hrs,which increases expression of HMG CoA reductase by four-fold aspreviously reported (15). Cells were incubated for another 6 hrsfollowing the addition of 25HC3S. Real time RT-PCR analysis shows that25HC3S still strongly inhibits HMG CoA reductase mRNA levels (FIG. 16B).Interestingly, 25HC shows much more potent inhibition of the reductasemRNA in lipid-depleted media as compared to cells cultured in mediumcontaining FBS (FIGS. 16A and 5B). Under these conditions, 25HC is morepotent inhibition than 25HC3S on HMG CoA reductase mRNA levels. Theresult of inhibition of HMG CoA reductase by 25HC is consistent withprevious report (16;17). To confirm its physiological role that 25HC3Splays in the cholesterol biosynthesis, primary human hepatocytes (PHH)cultured in serum-free media were used. Surprisingly, 25HC did notsignificantly affect the levels of HMG CoA reductase mRNA (˜15% at 25mM). In contract, 25HC3S inhibited HMG CoA reductase mRNA at a similarlevel (˜75%) as that in HepG2 cells (FIG. 16C). HPLC analysis showedthat the increasing levels of 25HC in the cells are concentrationdependent (FIGS. 17A-E), indicating that 25HC can still enter to thecells under this culture condition as in FBS containing media. Theseresults suggest that 25HC3S and 25HC inhibit HMG CoA reductase mRNA bydifferent mechanisms.

The Nuclear Oxysterol Inhibits HMG CoA Reductase Expression byInhibition of Both SREBP-1 and SREBP-2 Activation

It has been well documented that HMG CoA reductase gene expression isregulated by SREBP-1 and SREBP-2 (18). When SREBPs are activated, thecholesterol biosynthesis will increase (19). To study whether SREBPregulatory system is involved in the inhibition of HMG CoA reductasemRNA levels and inhibition of cholesterol biosynthesis by 25HC3S, totalcellular protein was extracted from HepG2 cells treated with 25HC3S or25HC at different concentration (FIG. 18). The precursor and matureforms of SREBPs were determined by Western blot analysis. As expected,the decreases of the mature forms of SREBP-1 and the increases of theprecursor form of SREBP-1 following addition of 25HC3S and 25HC weredose dependent (FIGS. 18A and 7B). However, the mature form of SREBP-2only slightly decreased (FIGS. 18A and 7B). It was observed that theactivation of SREBP-1 was much more sensitive to the treatment with 25HCand 25HC3S than that of SREBP-2. The inhibition of SREBP-1 maturationfits the decrease in HMG CoA reductase mRNA, at 3 μM of 25HC or 12 μM of25HC3S, 85% of the mRNA of HMG CoA reductase and SREBP 1 was inhibited.To confirm the mechanism, HepG2 cells were incubated in media containing50 μM of mevinolin and 0.5 μM of mevalonate. Under this condition,SREBPs and HMG CoA expressions are upregulated. Following the treatmentwith 25HC or 25HC3S, SREBPs activation in the cells was determined byWestern blot as described above. As shown in FIGS. 18C and 8D, 25HC3Sshows more potent inhibition on SREBP-1 activation than 25HC. Thus, thenuclear oxysterol, 25HC3S, is most likely to inhibit the activation ofSREBP-1 and subsequently inhibits the expression HMG CoA reductase.

Discussion

The current study shows that the chemically synthesized oxysterol,25HC3S as well as 25HC, inhibits the cholesterol biosynthesis and theexpression of the key enzyme, HMG CoA reductase. Unlike 25HC, 25HC3Sinhibits this expression in primary human hepatocytes as well as HepG2.25HC3S was found in the human liver nuclei and its levels weredramatically increased following overexpression of mitochondrialcholesterol delivery protein, StarD1, in primary rat hepatocytes,indicating that 25HC3 S is most likely synthesized in the mitochondriaand translocated to the nuclei for the regulation of gene expressioninvolved in cholesterol homeostasis (10). The present results providedevidence that the oxysterol, 25HC3S, plays an important role inmaintenance of the intracellular cholesterol homeostasis, and suggestedthat the StarD1 protein may serve as a sensor of intracellularcholesterol levels. When cholesterol levels are too high in the cells,StarD1 protein may deliver cholesterol to mitochondria where cholesterolis converted to be potent regulatory oxysterols such as 25HC, 27HC, and25HC3S. Those oxysterols play important roles in maintenance ofcholesterol homeostasis.

Theoretically, the added oxysterols can be metabolized after they enterinto cells: 25HC can be sulfated by hydroxysteroid sulfotransferase 2B1b(SULT2B1b) to be sulfated 25HC or sulfated 25HC can be degraded bysulfatase to be 25HC. Thus, which one serves as a potent regulator inthe maintenance of cholesterol homeostasis is questionable. Our HPLCanalysis data shows that no 25HC was generated following addition of25HC3S up to 50 μM (FIG. 17, left panels) suggesting that 25HC3S was notdegraded during the culture time. In contrast, the peak of 25HCincreased following the addition of the 25HC and the increasing is dosedependent (FIG. 17, right panels). Although the results can not rule outthe possibility that a small portion of the oxysterol was converted tobe the sulfated 25HC, there is no evidence that 25HC works as thesulfated form at present.

25HC3S and 25HC inhibit cholesterol synthesis by two differentmechanisms, both involving the proteins that control sterol regulatoryelement-binding proteins (SREBPs), membrane-bound transcription factorsthat activate genes encoding enyzymes of lipids biosynthesis. Insterol-depleted cells, SREBP cleavage-activating protein (SCAP) escortsits bound SREBP to the Golgi apparatus where the SREBP is processedsequentially by two membrane-embedded proteases. The NW-terminal domainreleased by the process can enter the nucleus where it activatestranscription of the gene encoding HMG CoA reductase and more than 30other genes whose products are necessary for lipid synthesis (18). When25HC is delivered to cells in ethanol or when cholesterol is deliveredin LDL, SCAP becomes trapped in the ER. The bound-SREBP is no longercarried to the Golgi apparatus, and the NH2-terminal domain can notenter the nucleus (20). As a result, transcription of the lipidbiosynthetic genes declines. Retention of the SCAP-SREBP complex in theER is mediated by the sterol-induced binding of SCAP to Insigs (Insig-1and Insig-2) in the ER membrane (20;21). When mixtures of cholesteroland 25HC are added to cultured cells, SCAP is induced to bind to Insig 1and Insig-2, and thus can not transport SREBPs to the Golgi body (3) andto be activated. J.L. Goldstein laboratory provided evidence thatcholesterol interacts with SCAP directly by inducing it to bind toInsigs, whereas 25HC works indirectly through a putative 25HC sensorprotein that elicits SCAP Insig binding (3). However, what the 25HCsensor protein is unknown.

The present study showed that 25HC strongly inhibits HMG CoA reductasein the presence of the lipids depleted serum both in HepG2 (FIG. 18)(˜90%), similar inhibits as 25HS3S does in the presence of serum (˜70%),but weakly inhibits in PHH when the cells were cultured in serum freemedia (FIG. 178) (˜15%). These results suggest that PHH does not express25-hydroxycholesteorl sensor protein(s) under this culture system.However, 25HC3S can inhibit the enzyme expression to similar levelseither presence or absence of serum or lipids depleted serum in bothhepG2 and PHH indicating that 25HC3 S can directly regulate the geneexpression. The major reason could be that 25HC3S is much morehydrophilic than 25-hydroxycholeterol and is water soluble, which makesthe molecule freely self-transport after this molecule enters the cells.

The oxysterol, 25HC3S, inhibits the cholesterol biosynthesis throughinhibiting the activation of SREBP-1 and SREBP-2, and subsequentlyinhibit the expression HMG CoA reductase in HepG2 cells and primaryhuman hepatocytes. Based on the microarray data from transgenic andknockout mice, both SREBP-1 and SREBP-2 activation stimulate HMG CoAreductase by 30 folds and 38 folds, respectively (18). SREBP-1 alsostrongly stimulates fatty acid synthase but not SREBP-2 (18). However,it is not clear whether SREBP-1 and SREBP-2 are directly mediated by25HC3S to regulate the expression regulation of the key enzyme, HMG CoA.Our results clearly show that 25HC3S as well as 25HC are much morepotent in inhibiting SREBP-1 activation than SREBP-2. We believe that25HC3S inhibits SREBP-1 activation and subsequently inhibits HMG CoAreductase resulting in the decline of cholesterol biosynthesis.

REFERENCES FOR EXAMPLE 4

-   1. Dubrac S, Lear S R, Ananthanarayanan M, Balasubramaniyan N,    Bollineni J, Shefer S et al. Role of CYP27A in cholesterol and bile    acid metabolism. J Lipid Res 2005; 46(1):76-85.-   2. Li X, Hylemon P, Pandak W M, Ren S. Enzyme activity assay for    cholesterol 27-hydroxylase in mitochondria. J Lipid Res 2006;    47(7):1507-1512.-   3. Adams C M, Reitz J, De Brabander J K, Feramisco J D, Li L, Brown    M S et al. Cholesterol and 25-hydroxycholesterol inhibit activation    of SREBPs by different mechanisms, both involving SCAP and Insigs. J    Biol Chem 2004; 279(50):52772-52780.-   4. Bjorkhem I, Diczfalusy U. Oxysterols: friends, foes, or just    fellow passengers? Arterioscler Thromb Vasc Biol 2002;    22(5):734-742.-   5. Corsini A, Verri D, Raiteri M, Quarato P, Paoletti R,    Fumagalli R. Effects of 26-aminocholesterol, 27-hydroxycholesterol,    and 25-hydroxycholesterol on proliferation and cholesterol    homeostasis in arterial myocytes. Arterioscler Thromb Vasc Biol    1995; 15(3):420-428.-   6. Fu X, Menke J G, Chen Y, Zhou G, Macnaul K L, Wright S D et al.    27-hydroxycholesterol is an endogenous ligand for liver X receptor    in cholesterol-loaded cells. J Biol Chem 2001; 276(42):38378-38387.-   7. Pandak W M, Ren S, Marques D, Hall E, Redford K, Mallonee D et    al. Transport of cholesterol into mitochondria is rate-limiting for    bile acid synthesis via the alternative pathway in primary rat    hepatocytes. J Biol Chem 2002; 277(50):48158-48164.-   8. Ren S, Hylemon P B, Marques D, Gurley E, Bodhan P, Hall E et al.    Overexpression of cholesterol transporter StAR increases in vivo    rates of bile acid synthesis in the rat and mouse. Hepatology 2004;    40(4):910-917.-   9. Hall E A, Ren S, Hylemon P B, Rodriguez-Agudo D, Redford K,    Marques D et al. Detection of the steroidogenic acute regulatory    protein, StAR, in human liver cells. Biochim Biophys Acta 2005;    1733(2-3):111-119.-   10. Ren S, Hylemon P, Zhang Z P, Rodriguez-Agudo D, Marques D, Li X    et al. Identification of a novel sulfonated oxysterol,    5-cholesten-3beta,25-diol 3-sulfonate, in hepatocyte nuclei and    mitochondria. J Lipid Res 2006; 47(5):1081-1090.-   11. Rodriguez-Agudo D, Ren S, Hylemon P B, Redford K, Natarajan R,    Del Castillo A et al. Human StarD5, a cytosolic StAR-related lipid    binding protein. J Lipid Res 2005; 46(8):1615-1623.-   12. Ren S, Scarsdale J N, Ariga T, Zhang Y, Klein R A, Hartmann R et    al. 0-acetylated gangliosides in bovine buttermilk. Characterization    of 7-O-acetyl, 9-O-acetyl, and 7,9-di-O-acetyl GD3. J Biol Chem    1992; 267(18):12632-12638.-   13. Hall E A, Ren S, Hylemon P B, Rodriguez-Agudo D, Redford K,    Marques D et al. Detection of the steroidogenic acute regulatory    protein, StAR, in human liver cells. Biochim Biophys Acta 2005;    1733(2-3):111-119.-   14. Laemmli U K. Cleavage of structural proteins during the assembly    of the head of bacteriophage T4. Nature 1970; 227(259):680-685.-   15. Hall E A, Ren S, Hylemon P B, Redford K, Del Castillo A, Gil G    et al. Mitochondrial cholesterol transport: a possible target in the    management of hyperlipidemia. Lipids 2005; 40(12):1237-1244.-   16. Panini S R, Sexton R C, Rudney H. Regulation of    3-hydroxy-3-methylglutaryl coenzyme A reductase by oxysterol    by-products of cholesterol biosynthesis. Possible mediators of low    density lipoprotein action. J Biol Chem 1984; 259(12):7767-7771.-   17. Sexton R C, Gupta A K, Panini S R, Rudney H. Progesterone    stimulation of HMG-CoA reductase activity in cultured cells. Biochim    Biophys Acta 1995; 1255(3):320-332.-   18. Horton J D, Shah N A, Warrington J A, Anderson N N, Park S W,    Brown M S et al. Combined analysis of oligonucleotide microarray    data from transgenic and knockout mice identifies direct SREBP    target genes. Proc Natl Acad Sci USA 2003; 100(21):12027-12032.-   19. Horton J D, Goldstein J L, Brown M S. SREBPs: activators of the    complete program of cholesterol and fatty acid synthesis in the    liver. J Clin Invest 2002; 109(9):1125-1131.-   20. Yang T, Espenshade P J, Wright M E, Yabe D, Gong Y, Aebersold R    et al. Crucial step in cholesterol homeostasis: sterols promote    binding of SCAP to INSIG-1, a membrane protein that facilitates    retention of SREBPs in ER. Cell 2002; 110(4):489-500.-   21. Yabe D, Xia Z P, Adams C M, Rawson R B. Three mutations in    sterol-sensing domain of SCAP block interaction with insig and    render SREBP cleavage insensitive to sterols. Proc Natl Acad Sci USA    2002; 99(26):16672-16677.

Example 5 In Vitro and In Vivo Testing of the Effect of the NuclearOxysterol 25HC3S on Triglyceride and Cholesterol Levels

The effect of the addition nuclear oxysterol on cholesterol levels wastested in human hepatocyte HepG2 cell line, and the results arepresented in FIG. 19. As can be seen, administration of the nuclearoxysterol resulted in a significant decrease in the level of cholesteroldetected in microsomal fractions of the HepG2 cells, when compared tothe control (25HC3S concentration=0).

Administration to female mice (20-25 g weight) were was by peritonealinjection once with or without 72 μg of the nuclear oxysterol in 100 μlof 0.9% NaCl containing 3 μl of ethanol. Twelve hrs following theinjection, blood was harvested for analysis of triglycerides andcholesterol. The results are presented in FIG. 20, which shows thatinjection of the nuclear oxysterol decreased triglycerides in serum by40% (n=12, p<0.01) and decreased cholesterol levels by 15%. Further,injection of the nuclear oxysterol did not increase the activities ofalkaline phosphotase, serum glutamate pyruvate transaminase (SGPT), andserum glutamic-oxaloacetic transaminase (SGOT) in sera suggesting thatthe nuclear oxysterol is non-toxic (data not shown).

To test the effect of diet on cholesterol levels, mice were providedwith either a normal or high cholesterol diet for 8 days, and treatedwith the nuclear oxysterol as described above. Liver tissues werecollected for pathohistochemistry studies (Sudan IV staining) and theresults showed that administration of the oxysterol significantlydecreased triglyceride levels in liver tissues of mice that were fednormally and also of mice that were fed a high cholesterol diet (datanot shown).

This study demonstrates that this natural nuclear oxysterol, 25HC3S,could serve as a potent drug for the treatment and prevention ofhypercholesterolemia and hyperlipidemia, and related diseases such asatherosclerosis.

Example 6 25-Hydroxycholesterol-3-Sulfate (25HC3S) Activates PPARγ andAttenuates Inflammatory Responses in Human Macrophages

The nuclear orphan receptor PPARγ is particularly important inregulating inflammatory responses in macrophages. Activation of PPARγrepresses key inflammatory response gene expressions. However, theregulation of PPARγ activation is obscure. Recently, a new cholesterolmetabolite, 25-hydroxycholesterol-3-sulfate (25HC3S), was identified asa potent regulatory molecule of lipid metabolism. This Example exploresthe effect of 25HC3S and its precursor, 25-hydroxycholesterol (25HC) onPPARγ activation and inflammatory responses. Addition of 25HC3S to humanmacrophages markedly increased nuclear PPARγ, cytosol IκB, and decreasednuclear NFκB protein levels. PPARγ response element reporter gene assaysshowed that 25HC3S significantly increased luciferase activities.NFκB-dependent promoter reporter gene assays showed that 25HC3Ssuppressed TNFα-induced luciferase activities only when co-transfectedwith pCMX-PPARγ plasmid in H441 cells. In addition, 25HC3S decreasedLPS-induced TNFα and IL-1β mRNA expressions and releases. In thePPARγ-specific siRNA transfected macrophages, 25HC3S failed to increaseIκB and to suppress TNFα and IL-1β expression. In contrast to 25HC3S,its precursor 25HC, a known LXR ligand, decreased nuclear PPARγ, cytosolIκB, while increasing nuclear NFκB protein levels in the presence of LPSand TNFα. This demonstrates that 25HC3S acts in macrophages as a PPARγactivator, and suppresses inflammatory response via PPARγ/IκB/NFκBsignaling pathway.

Macrophages are the key cellular players in the pathophysiology ofatherosclerosis. In the early stage of atherosclerosis, macrophages inarterial walls may accumulate lipids. These lipid-loaded macrophages,termed foam cells, are characteristic of a reversible early cellularphase of atherosclerotic lesions. Progressive lipid accumulation leadsto further escalation of inflammatory responses and infiltration ofinflammatory cells [1]. Through this process, early cellular lesions aretransformed to late, fibrous atherosclerotic plaques. Physiological orpharmacological maneuvers that reduce macrophage lipids and inflammatoryresponses may be effective in preventing or reversing atherosclerosis.

Nuclear orphan receptors are ligand-activated transcription factors thatregulate the expression of target genes to affect processes as diverseas reproduction, inflammation, development, and general metabolism [2].Nuclear receptor peroxisome proliferation activator receptors (PPARs),which play major roles in the regulation of lipid metabolism, glucosehomeostasis, and inflammatory process, are implicated in the control ofdiverse diseases such as type II diabetes and atherosclerosis, and arebelieved to be ideal targets for therapeutic management strategies formany metabolic disorders [3-8]. Activation of PPARγ leads to theformation of heterodimers with RXRs. The heterodimers bind toDNA-specific sequences called peroxisome proliferator response elements(PPRE) and lead to up- or down-regulation of transcriptional activity oftarget genes [9]. Several natural compounds such as fatty acids,PGD2-derivatives, eicosanoids have been reported to act on PPARγ[10-13]. However, their affinities with PPARγ protein are low [14-16].Thus, natural ligands for PPARγ and the regulating mechanisms of itsactivation are yet to be elucidated.

Oxygenated cholesterols, oxysterols, play an important role inmaintenance of lipid homeostasis [17]. Recently, we identified a noveloxysterol, 25-hydroxycholesterol-3 sulfate (25HC3S), that accumulates inhepatocyte nuclei following overexpression of the mitochondrialcholesterol delivery protein, StarD1 [18-20]. Macrophages are able tosynthesize this oxysterol [21]. This oxysterol appears to be synthesizedfrom 25HC by sterol sulfotransferase 2B1 (SULT2B1) [22]. Of note is thatoverexpression of SULT2B1 impairs the response of liver oxysterolreceptor LXR to multiple oxysterol ligands [23].

Furthermore, the addition of 25HC3S to primary hepatocytesdown-regulates expression of key enzymes involved in lipid metabolismand decreases lipid biosynthesis by inactivating the LXR/SREBP-1signaling pathway in hepatocytes and macrophages [21,24]. Severalstudies show that inflammation is closely associated with disorderedlipid metabolism [25,26]. Infection and inflammation induce theacute-phase response (APR), leading to multiple alterations in lipid andlipoprotein metabolism. APR increases plasma triglyceride levels, denovo hepatic fatty acid synthesis, and suppression of fatty acidoxidation [27]. The molecular mechanisms during the APR involvecoordinated changes in several nuclear orphan receptors, includingPPARs, LXRs, and RXRs [27,28]. PPARγ as well as LXRs are lipid-activatedtranscription factors and reciprocally regulate inflammation and lipidmetabolism [3,29]. The processes by which these events occur are poorlyunderstood. Cholesterol metabolites 25HC and 25HC3S have been shown tobe the potent regulators involved in lipids metabolism via LXRs/SREBPssignaling pathway [21,24]. It is thus possible that they are able toregulate inflammatory response through PPARγ signaling pathway.

Data presented in this Example demonstrates that 25HC3S is a potentdown-regulator while 25HC is an up-regulator of inflammatory responses.Evidence is provided that their effects on the inflammatory response aremediated via activation of the PPARγ/IκB/NFκB signaling pathway inmacrophages. These findings imply a new signaling pathway for theinteraction between inflammatory response and lipid metabolism.

Materials and Methods

Cell culture reagents and supplies were purchased from GIBCO BRL (GrandIsland, N.Y.); the reagents for real time RT-PCR were obtained from ABApplied Biosystems (Applied Biosystems, Foster City, Calif.). Antibodiesagainst human PPARγ, IκB, and Lamin B were purchased from Santa CruzBiotechnology (Santa Cruz, Calif.). FuGENE HD transfection reagent wasobtained from Roche Applied Science (Indianapolis, Ind.). Single AnalyteELISArray™ Kits were purchased from SupperArray (Frederick, Md.). TheDual-Glo Luciferase Assay System and pGL3-NFκB-luc were purchased fromPromega (Wisconsin, Wis.); PPAR agonist rosiglitazone and antagonistT0070907 were from New Cayman Chemical (Ann Arbor, Mich.). pGL3-PPARresponse element (PPRE)-luciferase reporter containing three copies ofPPRE from the promoter of rat acyl CoA oxidase, and the receptorexpression plasmids pcDNAI-PPAR were from the University of TennesseeHealth Science Center [30].

Cell Culture

Human THP-1 monocytes and H441 cells were purchased from the AmericanType Culture Collection (Manassas, Va.) and maintained according to thesupplier's protocols. THP-1 monocytes were differentiated to macrophagesby adding 100 nM of phorbol 12-myristate 13-acetate (PMA). When cellsreached ˜90% confluence, oxysterols in DMSO or in ethanol (the finalconcentration in media was <0.1%) were added as indicated. The cellswere harvested at the times as indicated. Nuclear and cytosolicfractions were isolated using NE-PER®, Nuclear and CytoplasmicExtraction Reagents (Pierce, Rockford, Ill. Western blot analysis ofnuclear PPAR and intracellular I B levels

Fifty μg of total cell lysates or nuclear protein extracts, otherwise asindicated, were separated on 10% SDS-PAGE gels and transferred onto apolyvinylidene difluoride membrane as described previously [31].Membranes were blocked in TBS containing 5% of nonfat dried milk for 1hr. The specific proteins were determined by incubation with specificantibodies against human PPAR or IκB overnight at 4° C. with shaking.After washing, the membrane was incubated in a 1:3,000 dilution of asecondary antibody (goat anti-rabbit or anti-mouse IgG-HP conjugate;Bio-Rad, Hercules, Calif.) at room temperature for 1 hr in the washingbuffer (Tris buffered solution containing 0.5% Tween 20). The proteinbands were visualized using Western Lightening Chemiluminescence Reagent(Perkin-Elmer, Waltham, Mass.). The expression levels were normalized toLamin.

PPARγ Transcription Factor Assay

PPAR response elements (PPRE) binding activities were measured using anenzyme-linked immunosorbent assay (PPAR transcription factor assay kit;Cayman Chemical, Ann Arbor, Mich.). The 96-well plate was preimmobilizedwith deoxyoligonucleoties containing PPRE. THP-1 derived macrophageswere treated with 25HC3S at indicated concentrations or rosiglitazonefor 4 hrs. The cells were then rinsed and nuclear proteins wereextracted according to the manufacturer's instructions. Total nuclearextract protein, 10 g from each sample, was added to the plate. The kitprovided two negative as zero controls, one positive as maximal bindingcontrol, and one competitive as specific binding control (in thepresence of dsDNA) were used in the assay. After incubating for 1 hr,the wells were washed and incubated with primary PPAR antibody whichrecognizes the accessible epitope on PPAR protein upon PPRE binding. Theperoxidase-labeled second antibody was added and incubated for 1 hr. Thereaction was stopped and absorbance was read at 450 nm in aspectrophotometer.

Transfection and Promoter Reporter Gene-Luciferase Assays

H441 cells were seeded in 96-well plates. When cell density reached90-95%, the cells were transfected with an expression plasmid asindicated using a lipid-based FuGENE HD transfection reagent accordingto the manufacture (Roche, Indianapolis, Ind.). A synthetic renillaluciferase reporter, phrG-TK (Promega, Wisconsin, Wis.), was used as aluciferase internal standard. For PPRE reporter gene assay, 50 ng ofpGL3-PPRE-acyl-CoA oxidase luciferase reporter, 50 ng of expressionplasmid pCMX-PPARγ, and 50 ng of phrG-TK vector (internal standard) werecotransfected as per the manufacture's instruction. Twenty four hrsafter the transfection, different concentration of 25HC3S,rosiglitazone, and/or T0070907 were added and incubated for another 24hrs. Luciferase activities were determined using the Dual-Glo LuciferaseAssay System according to the manufacturer's protocol (Promega,Wisconsin, Wis.). The amount of luciferase activity was measured using aTopCount NXT Microplate Scintillation and Luminescence Counter (Packard,Meriden, Conn.) and normalized to the amount of phG-TK luciferaseactivity. Transfections were carried out in triplicate for each sample,and each experiment was repeated three times.

Detection of Intracellular Distribution of PPARγ in THP-1 DerivedMacrophages

THP-1 derived macrophages were cultured on coverslips in six-well platesand treated with different concentrations of 25HC and 25HC3S for 4 hrs.The cells on coverslips were washed with PBS, fixed with 3.7%formaldehyde for 10 min at 4° C., and then rinsed three times with PBSat room temperature. They were then permeabilized with PBS containing0.1% of TritonX-100 for 3 min and washed with PBS before blocking byincubation with 5% of normal goat serum in PBS overnight at 4° C. Forinteraction with primary antibodies, cells were incubated with 2.5%normal goat serum in PBS containing PPARγ antibody for 1 hr in anincubator. Cells were washed in PBS containing 0.05% of Tween 20 (3×10min). The bound primary antibodies were visualized with Alexa Fluor 488goat anti-mouse IgG. The minor groove of double stranded DNA as anuclear marker was stained by DAPI. Afterwashing, coverslips weremounted on slides and viewed with a Zeiss LSM 510 Meta confocalmicroscope. Scalebar=10 μm.

Determination of mRNA Levels by Real-Time RT-PCR

Total RNA was isolated from THP-1 derived macrophages followingtreatments for 6 hrs otherwise as indicated using SV Total RNA IsolationKit (Promega, Wisconsin, Wis.), which includes DNase treatment. Twomicrogram of total RNA was used for first-strand cDNA synthesis asrecommended by manufacturer (Invitrogen, Carlsbad, Calif.). Real-timeRT-PCR was performed using SYBR Green as indicator on ABI 7500 FastReal-Time PCR System (Applied Biosystems, Foster City, Calif.). Thefinal reaction mixture contained 10 ng of cDNA, 100 nM of each primer,10 μl of 2×SYBR® Green PCR Master Mix (Applied Biosystems, Foster City,Calif.), and RNase-free water to complete the reaction mixture volume to20 All reactions were performed in triplicate. PCR was carried out for40 cycles of 95° C. for 15 s and 60° C. for 1 min. The fluorescence wasread during the reaction, allowing a continuous monitoring of the amountof PCR product. The data was normalized to internal control GAPDH. Thesequences of primers are used as recommended at the website located atpga.mgh.harvard.edu/primerband, as shown in Table 2.

TABLE 2 Primer sets used for real-time RT-PCR Gene GenBank Name Nos.Forward Sequence Reverse Sequence GAPDHR NM-002046 CAATGACCCCTTCATTGACCTTGATTTTGGAGGGATCTCG (SEQ ID NO: 3) (SEQ ID NO: 4) IL-1β NM-000576CACGATGCACCTGTACGATCA GTTGCTCCATATCCTGTCCCT (SEQ ID NO: 5) (SEQ ID NO: 6TNFα NM-000594 TCTTCTCGAACCCCGAGTGA CCTCTGATGGCACCACCAG (SEQ ID NO: 7)(SEQ ID NO: 8) IκB NM-001278 GGCTTCGGGAACGTCTGTC TGGCATGGTTCAACTTCTTCAT(SEQ ID NO: 9) (SEQ ID NO: 10) IL-8 NM-000584 ACTGAGAGTGATTGAGAGTGGACAACCCTCTGCACCCAGTTTTC (SEQ ID NO: 11) (SEQ ID NO: 12)

ELISA Analysis of Cytokine Releases

A total of 1×10⁶ macrophages were treated with LPS (1 μg/ml) and/ordifferent concentrations of 25HC or 25HC3S for 24 hrs. Supernatants wereharvested and cytokines IL-1B and TNF-α concentration were measured byenzyme-linked immunosorbent assay (ELISA) according to manufacturer'sinstruction (SuperArray Bioscience, Frederick Md.). SiRNA-mediatedmacrophage RNA interference

pSilencer2.1-U6 neo siRNA expression vector and negative control forRNAi were purchased from Ambion, Inc. (Austin, Tex.). Three human PPARγoligonucleotide sequences, GACTCAGCTCTACAATAAG (siRNA1, SEQ ID NO: 13),GCGATTCCTTCACTGATAC (siRNA2, SEQ ID NO: 14), and GCTTATCTATGACAGATGT(siRNA3, SEQ ID NO: 15), were selected as specific siRNAs to targethuman PPARγ. Synthetic sense and antisense oligonucleotides wereannealed by incubated at 90° C. for 3 min and then at 37° C. for 1 hr.The double stranded oligonucleotides were cloned into the BamH I-HindIII sites of the pSilencer2.1-U6 neo vector according manufacturer'sprotocol. The control RNA interference (RNAi) sequence was a randomlyscrambled and was not found in the mouse, human, or rat genomedatabases. All of the constructs were confirmed by sequencing. THP-1macrophages were transfected with a PPARγ siRNA or control RNAi usingFugeneHD reagent according to the manufacturer's instructions (RocheApplied Science, Indianapolis, Ind.). After incubation for 4 hrs, themedium was changed with normal medium and compounds were added atappropriate concentrations as indicated. Cells were harvested after 48hrs following the addition, and PPARγ protein levels were determinedusing western blot and mRNA levels of inflammatory response factors weremeasured by real time RT-PCR analysis.

Statistics

Data are reported as mean±standard deviation (SD). Where indicated, datawere subjected to t-test or ANOVA analysis and determined to besignificantly different at p<0.05.

Results 25HC3S Administration Increases Nuclear PPARγ Levels inMacrophages

To examine the effect of 25HC3S and its precursor 25HC on PPARγactivation, total nuclear proteins were extracted and nuclear PPARγlevels were determined by western blot analysis. As shown in FIG. 21,addition of 25HC3S to the macrophages led to significant concentration-(FIG. 21A, upper panels) and time- (data not shown) dependent increasesin nuclear PPARγ protein levels. To confirm the increasing 52 kDa bandwas PPARγ protein, an artificial PPARγ ligand, rosiglitazone, was usedas positive control and a specific antagonist, T0070907, as negativecontrol (FIG. 21B). As shown in FIG. 21A, 25HC3S and rosiglitazonesubstantially increased nuclear PPARγ levels (Lane R) while T0070907decreased the level (Lane T). In contrast, 25HC decreased PPARγ levels(middle figures of FIG. 21A). It was noticed that the increases ordecreases in the nuclear PPARγ levels occurred only in the early stages,less than 4 hrs (data not shown). Summaries of theconcentration-dependent increases or decreases after normalization tonuclear protein lamin are shown in the lower panel of FIG. 21A. Theseresults suggest that 25HC3S functions as a PPARγ agonist and 25HC as anantagonist. To further confirm that 25HC3S binds with the same moleculeas the artificial antagonist, a competitive assay was performed. In thepresence of the antagonist T0070907, 25HC3S failed to increase thenuclear PPARγ levels to its maxima. However, the levels of inhibitioncould be partially reversed following increasing concentration of 25HC3Sas shown in the top panel of FIG. 21B. In contrast, rosiglitazoneincreased nuclear PPARγ levels and the increased levels could besignificantly inhibited by the presence of 25HC as shown in the middlepanel of FIG. 21B. These results suggested that 25HC3S/T0070907 and25HC/rosiglitazone are competitive and they bind with the same molecule,PPARγ. Real time RT-PCR analysis showed that neither 25HC nor 25HC3S wasable to change PPARγ mRNA levels significantly (data not shown),suggesting these oxysterols have no effect on its transcriptionalregulation.

Double immunofluorescence studies using DAPI for the nuclear markertogether with the PPARγ antibody showed that PPARγ proteins were widelydistributed in the cytosols and nuclei of the macrophages (FIGS. 27A-F):compared with DMSO, administration of 25HC3S increased the nuclear PPARγfluorescence intensities; administration of the antagonist T0070907decreased the intensity, and blunted 25HC3S stimulation. The resultsimply that 25HC3S increases PPARγ levels in the nuclei by increasingnuclear translocation.

25HC3S Addition Increases PPARγ-Response Transcriptional Activities andRepresses Inflammatory Responses

To study the transcriptional activities of the nuclear extracts from25HC3S-treated macrophages, PPARγ responsive elements (PPRE) immobilizedELISA and PPRE reporter gene assay were carried out as shown in FIG. 22.ELISA assay showed that addition of 25HC3S significantly increased thePPRE-binding activities of the nuclear extracts, which isconcentration-dependent (p<0.01) (FIG. 22A). PPRE reporter gene assayswere performed in H441 cells because our preliminary experiments showedthat these cells had the best transfection efficiency and highestreciferase activities (data not shown). At 25 μM of 25HC3S, the activityreached to a similar levels as that induced by the PPARγ agonistrosiglitazone. In the presence of the antagonist T0070907, 25HC3S failed(p<0.01) to increase PPARγ reporter gene activities withco-overexpression of ACOX-PPRE reporter and PPARγ genes (FIG. 22B).Interestingly, in the presence of T0070907, the lower concentrations ofrosiglitazone failed to stimulate the reporter gene activity (p<0.01)but the higher concentrations still could increase the activities asshown in FIG. 22C. The results suggested that 25HC3S activates PPARγ bybinding with different motif and rosiglitazone binds with the same motifof the molecule as T0070907 or with different affinities. Addition of25HC3S suppresses LPS-induced IL-1β and TNFα expressions and releases inmacrophages. Addition of LPS substantially increased IL-1β mRNA levelsby 55-fold and significantly increased TNFα by 2-fold as previouslyreported [32,33]. Addition of 25HC3S suppressed LPS-induced IL-1β mRNAlevels by 2-fold while 25HC increased its levels by 2-fold (FIGS. 28Aand B). However, both 25HC3S and 25HC repressed LPS-induced TNFα mRNAlevels by two and four folds, respectively (FIGS. 28A and B). It wasnoticed that 25HC was significantly stronger than 25HC3S in suppressingLPS-induced TNFα expression (p<0.05). The suppressions of IL-1β and TNFαexpression by 25HC3S could significantly be blunted in the presence ofartificial PPARγ antagonist T0070907 (p<0.05) as shown in FIG. 22D.These results suggested that 25HC3S inhibits LPS-induced IL-1β and TNFαexpression by PPARγ signaling pathway.

Consistently, 25HC3S represses the LPS-induced releases of IL-1β andTNFα in the macrophages. Addition of LPS increased TNFα release by50-fold (data not shown) and increased IL-1β releases by 10-fold (FIG.22E, Lanes C and L), which was similar with previous reports [32,33].Interestingly, addition of 25HC3S significantly decreased LPS-inducedIL-1β release, but 25HC increased IL-1β release (p<0.05), which areconcentration-dependent manner as shown in FIG. 22E.

PPARγ is Involved in 25HC3S-Mediated Suppression of InflammatoryResponses Via NFκB

TNFα can activate IκB kinase, which phosphorylates IκB followed by itsubiquitination and subsequent degradation. In the absence of TNFα 25HC3Streatment decreased (p<0.05) but 25HC increased nuclear NFκB levels(Left panels in FIG. 23A). In the presence of TNFα, 25HC3S failed todecrease, but 25HC still significantly increased its nuclear NFκB levels(Right panels in FIG. 23A). In its inactive form, NFκB is sequestered inthe cytoplasm, bound by members of the IκBs. When IκBs arephosphorylated, ubiquitinated, and degraded, NFκB will be activated andenter nuclei. Thus, the results imply that the decreases/increases inthe nuclear NFκB by these oxysterols are related with IκB degradation.

To investigate whether the NFκB regulation is PPARγ dependent, H441cells were transfected with pNFκB dependent reporter gene-Luc expressionplasmid alone or co-transfected with PPARγ expression plasmid. In theabsence of PPARγ expression plasmid, TNFα induced the reporter geneexpression by 10-fold and 25HC3S failed to suppress its induction asshown in left panel of FIG. 23B. In the co-transfected cells, thepresence of PPARγ expression, TNFα still induced the reporter geneexpression by 10-fold but 25HC3S reduced its induction by 50% (Rightpanel of FIG. 23B). Furthermore, in the presence of PPARγ antagonist,the suppression was blunted (Right panel of FIG. 23B). The resultssuggested that the suppression of TNFα-induced NFκB dependent reportergene expression by 25HC3S requires the presence of PPARγ protein.

To confirm that the suppression of LPS-induced TNFα and IL-1β expressionis PPARγ dependent, specific siRNAs were used to knock-down PPARγ.Following transfection of the recombinant plasmid encoding specificsiRNAs for 48 hrs, about 45% of the cells were viable and harvested.About 90% of PPARγ protein levels were suppressed and their levels werenot changed in the presence of 12 μM of 25HC3S as shown in FIG. 24A.Consistently, 25HC3S significantly increased IκB mRNA levels by 2.5folds, and in the presence of TNFα, 25HC3S increased the levels by 6folds, which can be abolished by PPARγ specific siRNA (FIG. 24B). Asexpected, LPS stimulated TNFα by 40 folds and IL-1β expression by 12folds, and 12 μM of 25HC3S significantly suppressed LPS-induced TNFα(FIG. 24C), IL-1β (FIG. 24D), and NFκB (FIG. 24E) expression (p<0.05).However, in the siRNA-expressed cells, 25HC3 S failed in suppressingthese expressions (FIGS. 24C, 24D and 24E). These results suggest thatthe 25HC3S induces repression of nuclear NFκB levels and subsequentlydecreases TNFα and IL-1β expression through PPARγ induced IκBexpression.

To further confirm that 25HC3S/25HC regulates inflammatory responsesthrough NFκB signaling pathway, the effects of these two oxysterols onIκB expression were examined. When the macrophages were treated with25HC3S, the expression IκB at protein and mRNA levels were significantlyincreased (FIGS. 25A and 25B). Western blot analysis showed that 25HC3Sincreased and 25HC decreased IκB protein levels (Upper panel of FIGS.25A and 25B). When treated with 25HC, the mRNA level had nosignificantly changed (p>0.05 by ANOVA analysis) (FIG. 25C) but theprotein levels were significantly concentration-dependent decreased(p<0.05 by ANOVA analysis) (Lower panel of FIGS. 25A and 25B),indicating 25HC increases IκB protein degradation. In the presence ofTNFα, 25HC3S increased IκB protein levels more than those in the absence(Middle panel, FIGS. 25A and 25B), but in the presence of PPARγantagonist T0070907, 25HC3S failed to increase its levels (data notshown). These results indicated that PPARγ/IκB/NFκB signaling pathway isinvolved in 25HC3S/25HC regulated inflammatory responses.

Discussion

Previous reports show that 25HC and 25HC3S serve as ligands, agonist andantagonist, respectively of LXR nuclear receptor [21, 24]. In thepresent study, we have shown that 25HC3S, increases nuclear PPARγ,cytosol IκB, and decreases nuclear NF?B protein levels; increases PPARγtranscriptional activities and IκB expression; subsequently inhibits theTNFα and LPS-induced inflammatory factor expressions and releases inhuman THP-1 derived macrophages. In contrast, its precursor 25HC, aknown LXR ligand, basically has an opposite function. Thus, the presentresults provide strong evidence that 25HC and 25HC3S not onlycoordinately regulate lipid metabolism by LXR/SREBP-1 [21,24], but alsoinflammatory responses by PPARγ IκB/NFκB signaling pathway [46]. Therole of oxysterols including 25HC in inflammation is controversial. Thenuclear receptors LXR and PPARγ regulate inflammation in different waysand respond to distinct signaling pathways [3]. PPARs and LXRs bothexert positive and negative control over the expression of a range ofmetabolic and inflammatory genes. Although LXRs as well as PPARγ cantransrepress several inflammatory genes in a similar manner, comparativeDNA-microarray studies have identified overlapping, but distinct,subsets of genes that are repressed by ligand binding [34-37]. Why thesenuclear receptors use parallel molecular mechanisms to negativelyregulate similar but distinct gene subsets in the same cell type remainsan open and intriguing question [38, 39]. In our previous publication,we showed that 25HC is a potent agonist of LXR as others previouslydescribed, but 25HC3S is a potent antagonist [21]. In the present studywe have shown that 25HC is a potent PPARγ antagonist and 25HC3S is apotent agonist. Thus, both of the oxysterols can regulate inflammatoryresponses but via two different pathways. 25HC can suppress inflammatoryresponse via LXR signaling pathway but stimulate the response via PPARγpathway; 25HC3S can suppress inflammatory response via PPARγ pathway butmay interfere in a different direction by the LXR pathway. Severallaboratories found that oxysterols including 25HC can activate LXRs,subsequently repress a set of inflammatory genes after LPS and cytokinesstimulation [40, 41]. However, many other studies found that oxysterolsincluding 25HC induce inflammation and oxidation in different kinds ofcells. For example, 25HC substantially increased the IL-1β mRNAexpression and secretion induced by LPS in human monocyte-derivedmacrophages. 25HC is also a potent inducer of MCP-1, MIP-1β and IL-8secretion in vitro [41, 42]. 25HC treatments result in a significantincrease in NFκB transcriptional activity, not only by affecting the IκBdegradation and the translocation of p65/NFκB to the nucleus, but alsoby regulating the p65/NFκB transactivation [43]. Oxysterols also induceinflammation and oxidation through inducing slight mitochondrialdysfunctions and increasing reactive oxygen species (ROS) [43]. Previousstudy showed that PPARγ agonists increase LXR and ABCA1 mRNA levelsfollowing incubation with the agonists for 24 hrs [44]. It was assumedthat PPARγ regulates inflammatory response through LXR pathway. However,if the cells were incubated with the agonists for short times (less than6 hrs), the results were completely different [21]. Previous studieshave shown that 25HC3S decreases LXR activities and its targeting geneexpressions in macrophages. In contrast, LXR ligand, 25HC, increasesnuclear LXR levels following short incubation time, subsequentlyincreases LXR targeting gene expressions, inclusive of ABCA1/G1 andSREBP-1 mRNAs by 5-50-fold in 6 hrs. Furthermore, 25HC3S blocked thestimulation of target gene expressions induced by 25HC or artificial LXRagonist. It was concluded that 25HC3S activates PPARγ and modulatesinflammatory response through PPARγ not LXR signaling pathway in themacrophages. The present studies provide a new clue of the role thatoxysterols and oxysterol sulfation may play in inflammation regulationvia activation/inactivation of the nuclear orphan receptors such as LXRsand PPARs.

Previous studies have shown that PPARγ suppresses target gene expressionof NFB, nuclear factor of activated T cells (NFAT), activator protein-1(AP-1), and signal transducers and activator of transcription (STATS) inresponse to a variety of inflammatory stimuli, including cytokines andTLR ligands [45]. The mechanisms involved in the repressive effects ofPPARγ have yet to be elucidated. In the present study, it has been foundthat 25HC3S increases PPARγ and decreases NFB protein levels in nuclei(FIGS. 21 and 23); suppresses the expression of TNF-induced NFBdependent reporter gene, which is PPARγ-dependent (FIG. 24); induces IBexpression (FIG. 25); and inhibits IL-1β and TNFα expression (FIG. 22).It is possible that 25HC3S activates PPARγ, which induces IκB expressionand inhibits IκB degradation by suppressing of TNF expression. IκBprotein inhibits inflammatory responses by binding and inactivating NFB.Meanwhile, the activated PPARγ inhibits TNFα expression, which directlydecreases IB ubiquitination and degradation. Thus, 25HC3S attenuates theinflammatory response by increasing IκB expression and decreasing IBubiquitination and degradation through PPARγ/IB/NFB signaling pathway asshown in FIG. 26.

REFERENCES FOR EXAMPLE 6

-   1. B. Geeraert, K. D. De, P. C. Davey, F. Crombe, N. Benhabiles,    and P. Holvoet, Oxidized low-density lipoprotein-induced expression    of ABCA1 in blood monocytes precedes coronary atherosclerosis and is    associated with plaque complexity in hypercholesterolemic pigs, J.    Thromb. Haemost. 5 (2007) 2529.-   2. A. Chawla, J. J. Repa, R. M. Evans, and D. J. Mangelsdorf,    Nuclear receptors and lipid physiology: opening the X-files, Science    294 (2001) 1866.-   3. S. J. Bensinger and P. Tontonoz, Integration of metabolism and    inflammation by lipid-activated nuclear receptors, Nature 454 (2008)    470.-   4. M. A. Bouhlel, B. Derudas, E. Rigamonti, R. Dievart, J.    Brozek, S. Haulon, C. Zawadzki, B. Jude, G. Torpier, N. Marx, B.    Staels, and G. Chinetti-Gbaguidi, PPARgamma activation primes human    monocytes into alternative M2 macrophages with anti-inflammatory    properties, Cell Metab 6 (2007) 137.-   5. A. Castrillo and P. Tontonoz, PPARs in atherosclerosis: the clot    thickens, J. Clin. Invest 114 (2004) 1538.-   6. A. Chawla, W. A. Boisvert, C. H. Lee, B. A. Laffitte, Y.    Barak, S. B. Joseph, D. Liao, L. Nagy, P. A. Edwards, L. K.    Curtiss, R. M. Evans, and P. Tontonoz, A PPAR gamma-LXR-ABCA1    pathway in macrophages is involved in cholesterol efflux and    atherogenesis, Mol. Cell 7 (2001) 161.-   7. M. I. Dushkin, O. M. Khoshchenko, E. N. Posokhova, and Y. S.    Schvarts, Agonists of PPAR-alpha, PPAR-gamma, and RXR inhibit the    formation of foam cells from macrophages in mice with inflammation,    Bull. Exp. Biol. Med. 144 (2007) 713.-   8. J. I. Odegaard, R. R. Ricardo-Gonzalez, M. H. Goforth, C. R.    Morel, V. Subramanian, L. Mukundan, A. R. Eagle, D. Vats, F.    Brombacher, A. W. Ferrante, and A. Chawla, Macrophage-specific    PPARgamma controls alternative activation and improves insulin    resistance, Nature 447 (2007) 1116.-   9. W. Ahmed, O. Ziouzenkova, J. Brown, P. Devchand, S. Francis, M.    Kadakia, T. Kanda, G. Orasanu, M. Sharlach, F. Zandbergen, and J.    Plutzky, PPARs and their metabolic modulation: new mechanisms for    transcriptional regulation?, J. Intern. Med. 262 (2007) 184.-   10. H. Martin, Role of PPAR-gamma in inflammation. Prospects for    therapeutic intervention by food components, Mutat. Res. 669 (2009)    1.-   11. L. Villacorta, F. J. Schopfer, J. Zhang, B. A. Freeman,    and Y. E. Chen, PPARgamma and its ligands: therapeutic implications    in cardiovascular disease, Clin. Sci. (Lond) 116 (2009) 205.-   12. O. Nosjean and J. A. Boutin, Natural ligands of PPARgamma: are    prostaglandin J(2) derivatives really playing the part?, Cell    Signal. 14 (2002) 573.-   13. J. Berger and D. E. Moller, The mechanisms of action of PPARs,    Annu. Rev. Med. 53 (2002) 409.-   14. C. Yu, L. Chen, H. Luo, J. Chen, F. Cheng, C. Gui, R. Zhang, J.    Shen, K. Chen, H. Jiang, and X. Shen, Binding analyses between Human    PPARgamma-LBD and ligands, Eur. J. Biochem. 271 (2004) 386.-   15. M. J. DeGrazia, J. Thompson, J. P. Heuvel, and B. R. Peterson,    Synthesis of a high-affinity fluorescent PPARgamma ligand for    high-throughput fluorescence polarization assays, Bioorg. Med. Chem.    11 (2003) 4325.-   16. O. Nosjean and J. A. Boutin, Natural ligands of PPARgamma: are    prostaglandin J(2) derivatives really playing the part?, Cell    Signal. 14 (2002) 573.-   17. S. Gill, R. Chow, and A. J. Brown, Sterol regulators of    cholesterol homeostasis and beyond: the oxysterol hypothesis    revisited and revised, Prog. Lipid Res. 47 (2008) 391.-   18. W. M. Pandak, S. Ren, D. Marques, E. Hall, K. Redford, D.    Mallonee, P. Bohdan, D. Heuman, G. Gil, and P. Hylemon, Transport of    cholesterol into mitochondria is rate-limiting for bile acid    synthesis via the alternative pathway in primary rat hepatocytes, J.    Biol. Chem. 277 (2002) 48158.-   19. S. Ren, P. B. Hylemon, D. Marques, E. Gurley, P. Bodhan, E.    Hall, K. Redford, G. Gil, and W. M. Pandak, Overexpression of    cholesterol transporter StAR increases in vivo rates of bile acid    synthesis in the rat and mouse, Hepatology 40 (2004) 910.-   20. S. Ren, P. Hylemon, Z. P. Zhang, D. Rodriguez-Agudo, D.    Marques, X. Li, H. Zhou, G. Gil, and W. M. Pandak, Identification of    a novel sulfonated oxysterol, 5-cholesten-3beta,25-diol 3-sulfonate,    in hepatocyte nuclei and mitochondria, J. Lipid Res. 47 (2006) 1081.-   21. Y. Ma, L. Xu, D. Rodriguez-Agudo, X. Li, D. M. Heuman, P. B.    Hylemon, W. M. Pandak, and S. Ren, 25-Hydroxycholesterol-3-sulfate    regulates macrophage lipid metabolism via the LXR/SREBP-1 signaling    pathway, Am. J. Physiol Endocrinol. Metab 295 (2008) E1369-E1379.-   22. X. Li, W. M. Pandak, S. K. Erickson, Y. Ma, L. Yin, P. Hylemon,    and S. Ren, Biosynthesis of the regulatory oxysterol, 5-cholesten-3    {beta},25-diol 3-sulfate, in hepatocytes, J. Lipid Res. 48 (2007)    2587.-   23. W. Chen, G. Chen, D. L. Head, D. J. Mangelsdorf, and D. W.    Russell, Enzymatic reduction of oxysterols impairs LXR signaling in    cultured cells and the livers of mice, Cell Metab 5 (2007) 73.-   24. S. Ren, X. Li, D. Rodriguez-Agudo, G. Gil, P. Hylemon, and W. M.    Pandak, Sulfated oxysterol, 25HC3S, is a potent regulator of lipid    metabolism in human hepatocytes, Biochem. Biophys. Res. Commun.    360 (2007) 802.-   25. M. K. Heliovaara, A. M. Teppo, S. L. Karonen, and P. Ebeling,    Inflammation affects lipid metabolism during recovery from    hyperinsulinaemia, Eur. J. Clin. Invest 36 (2006) 860.-   26. T. P. Erlinger, E. R. Miller, III, J. Charleston, and L. J.    Appel, Inflammation modifies the effects of a reduced-fat    low-cholesterol diet on lipids: results from the DASH-sodium trial,    Circulation 108 (2003) 150.-   27. W. Khovidhunkit, M. S. Kim, R. A. Memon, J. K. Shigenaga, A. H.    Moser, K. R. Feingold, and C. Grunfeld, Effects of infection and    inflammation on lipid and lipoprotein metabolism: mechanisms and    consequences to the host, J. Lipid Res. 45 (2004) 1169.-   28. K. A. von, M. Soller, and B. Brune, Peroxisome    proliferator-activated receptor gamma (PPAR gamma) and sepsis, Arch    Immunol. Ther. Exp. (Warsz.) 55 (2007) 19.-   29. A. Castrillo and P. Tontonoz, Nuclear receptors in macrophage    biology: at the crossroads of lipid metabolism and inflammation,    Annu. Rev. Cell Dev. Biol. 20 (2004) 455.-   30. C. Zhang, D. L. Baker, S. Yasuda, N. Makarova, L. Balazs, L. R.    Johnson, G. K. Marathe, T. M. McIntyre, Y. Xu, G. D.    Prestwich, H. S. Byun, R. Bittman, and G. Tigyi, Lysophosphatidic    acid induces neointima formation through PPARgamma activation, J.    Exp. Med. 199 (2004) 763.-   31. S. Ren, P. Hylemon, D. Marques, E. Hall, K. Redford, G. Gil,    and W. M. Pandak, Effect of increasing the expression of cholesterol    transporters (StAR, MLN64, and SCP-2) on bile acid synthesis, J.    Lipid Res. 45 (2004) 2123.-   32. J. Q. Jin, C. Q. Li, and L. C. He, Down-regulatory effect of    usnic acid on nuclear factor-kappaB-dependent tumor necrosis    factor-alpha and inducible nitric oxide synthase expression in    lipopolysaccharide-stimulated macrophages RAW 264.7, Phytother. Res.    (2008).-   33. H. J. Hwang, H. J. Lee, C. J. Kim, I. Shim, and D. H. Hahm,    Inhibitory Effect of Amygdalin on Lipopolysaccharide-inducible    TNF-alpha and IL-1beta mRNA Expression and Carrageenan-induced Rat    Arthritis, J. Microbiol. Biotechnol. 18 (2008) 1641.-   34. F. Morello, E. Saglio, A. Noghero, D. Schiavone, T. A.    Williams, A. Verhovez, F. Bussolino, F. Veglio, and P. Mulatero,    LXR-activating oxysterols induce the expression of inflammatory    markers in endothelial cells through LXR-independent mechanisms,    Atherosclerosis 207 (2009) 38.-   35. D. Torocsik, A. Szanto, and L. Nagy, Oxysterol signaling links    cholesterol metabolism and inflammation via the liver X receptor in    macrophages, Mol. Aspects Med. 30 (2009) 134.-   36. C. Joffre, L. Leclere, B. Buteau, L. Martine, S. Cabaret, L.    Malvitte, N. Acar, G. Lizard, A. Bron, C. Creuzot-Garcher, and L.    Bretillon, Oxysterols induced inflammation and oxidation in primary    porcine retinal pigment epithelial cells, Curr. Eye Res. 32 (2007)    271.-   37. C. J. Delvecchio, P. Bilan, K. Radford, J. Stephen, B. L.    Trigatti, G. Cox, K. Parameswaran, and J. P. Capone, Liver X    receptor stimulates cholesterol efflux and inhibits expression of    proinflammatory mediators in human airway smooth muscle cells, Mol.    Endocrinol. 21 (2007) 1324.-   38. A. Vejux, L. Malvitte, and G. Lizard, Side effects of    oxysterols: cytotoxicity, oxidation, inflammation, and    phospholipidosis, Braz. J. Med. Biol. Res. 41 (2008) 545.-   39. I. Bjorkhem and U. Diczfalusy, Oxysterols: friends, foes, or    just fellow passengers?, Arterioscler. Thromb. Vasc. Biol. 22 (2002)    734.-   40. F. Morello, E. Saglio, A. Noghero, D. Schiavone, T. A.    Williams, A. Verhovez, F. Bussolino, F. Veglio, and P. Mulatero,    LXR-activating oxysterols induce the expression of inflammatory    markers in endothelial cells through LXR-independent mechanisms,    Atherosclerosis (2009).-   41. T. Rosklint, B. G. Ohlsson, O. Wiklund, K. Noren, and L. M.    Hulten, Oxysterols induce interleukin-1beta production in human    macrophages, Eur. J. Clin. Invest 32 (2002) 35.-   42. C. Prunet, T. Montange, A. Vejux, A. Laubriet, J. F.    Rohmer, J. M. Riedinger, A. Athias, S. Lemaire-Ewing, D. Neel, J. M.    Petit, E. Steinmetz, R. Brenot, P. Gambert, and G. Lizard,    Multiplexed flow cytometric analyses of pro- and anti-inflammatory    cytokines in the culture media of oxysterol-treated human monocytic    cells and in the sera of atherosclerotic patients, Cytometry A    69 (2006) 359.-   43. L. Calleros, M. Lasa, M. J. Toro, and A. Chiloeches, Low cell    cholesterol levels increase NFkappaB activity through a p38    MAPK-dependent mechanism, Cell Signal. 18 (2006) 2292.-   44. G. Chinetti, S. Lestavel, V. Bocher, A. T. Remaley, B.    Neve, I. P. Torra, E. Teissier, A. Minnich, M. Jaye, N.    Duverger, H. B. Brewer, J. C. Fruchart, V. Clavey, and B. Staels,    PPAR-alpha and PPAR-gamma activators induce cholesterol removal from    human macrophage foam cells through stimulation of the ABCA1    pathway, Nat. Med. 7 (2001) 53.-   45. C. K. Glass and S. Ogawa, Combinatorial roles of nuclear    receptors in inflammation and immunity, Nat. Rev. Immunol. 6 (2006)    44.-   46. S. Ren, L. Xu, P. Hylemon, D. Heuman, W M. Pandak,    25-Hydroxycholesterol and 25-hyddoxycholesterol 3-sulfate    reciprocally regulate lipid metabolism and inflammation in    hepatoryctes and macrophages. The 60th Annual Meeting of the    American Association for the Study of Liver Diseases, Boston, Mass.,    Oct. 30-Nov. 3, (2009), Hepatology 55 (4) (2009)16.

While the invention has been described in terms of its preferredembodiments, those skilled in the art will recognize that the inventioncan be practiced with modification within the spirit and scope of theappended claims. Accordingly, the present invention should not belimited to the embodiments as described above, but should furtherinclude all modifications and equivalents thereof within the spirit andscope of the description provided herein.

1-11. (canceled)
 12. A compound of the formula:

wherein the compound is in solid form.
 13. The compound of claim 12,wherein said compound is a pharmaceutically acceptable salt.
 14. Thecompound of claim 13, wherein said pharmaceutically acceptable salt is asodium salt.
 15. The compound of claim 12, wherein the compound is insubstantially purified form.
 16. The compound of claim 12, wherein thecompound is in a form that is at least about 80% free from otherchemical species.
 17. The compound of claim 12, wherein the compound isin a form that is at least about 90% free from other chemical species.18. The compound of claim 12, wherein the compound is in a form that isat least about 95% free from other chemical species.
 19. The compound ofclaim 13, wherein the compound is in substantially purified form. 20.The compound of claim 13, wherein the compound is in a form that is atleast about 80% free from other chemical species.
 21. The compound ofclaim 13, wherein the compound is in a form that is at least about 90%free from other chemical species.
 22. The compound of claim 13, whereinthe compound is in a form that is at least about 95% free from otherchemical species.
 23. The compound of claim 14, wherein the compound isin substantially purified form.
 24. The compound of claim 14, whereinthe compound is in a form that is at least about 80% free from otherchemical species.
 25. The compound of claim 14, wherein the compound isin a form that is at least about 90% free from other chemical species.26. The compound of claim 14, wherein the compound is in a form that isat least about 95% free from other chemical species.
 27. The compound ofclaim 12, wherein the compound is in powder form.
 28. A method of makinga compound of the formula:

comprising sulfating a 3β position of 25-hydroxycholesterol.
 29. Themethod of claim 28, wherein said sulfating is performed by forming amixture of said 25-hydroxycholesterol with sulfur trioxide triethylamine complex and incubating said mixture under conditions whereby said3β position of said 25-hydroxycholesterol is sulfated.
 30. The method ofclaim 28, further comprising purifying said compound by flashchromatography.
 31. The method of claim 28, further comprising purifyingsaid compound by HPLC.